Manipulation of microparticles in microfluidic systems

ABSTRACT

An array of transportable particle sets is used in a microfluidic device for performing chemical reactions in the microfluidic device. The microfluidic device comprises a main channel and intersecting side channels, the main channel and side channels forming a plurality of intersections. The array of particle sets is disposed in the main channel, and the side channels are coupled to reagents. As the particle sets are transported through the intersections of the main channel and the side channels, reagents are flowed through the side channels into contact with each array member (or selected array members), thereby providing a plurality of chemical reactions in the microfluidic system.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/928,808 (now abandoned), filed Oct. 30, 2007, which is a divisionalof U.S. patent application Ser. No. 10/606,201 (now abandoned), filedJun. 25, 2003, which is a continuation of U.S. patent application Ser.No. 09/510,626, filed Feb. 22, 2000, now U.S. Pat. No. 6,632,655, whichclaims benefit of and priority to U.S. Ser. No. 60/121,223, filed Feb.23, 1999 by Mehta et al.; U.S. Ser. No. 60/127,825, filed Apr. 5, 1999,by Mehta et al.; U.S. Ser. No. 60/128,643, filed Apr. 9, 1999, by Mehtaet al.; co-filed PCT application PCT/US00/04522, filed Feb. 22, 2000;co-filed U.S. application U.S. Ser. No. 09/510,205 (now U.S. Pat. No.6,613,513), filed Feb. 22, 2000; and co-filed PCT applicationPCT/US00/04486, filed Feb. 22, 2000. Each of these applications isincorporated herein by reference in its entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The present invention was made with government funding from the UnitedStates National Institute of Standards and Technology (NIST), throughthe Advanced Technology Program (ATP) under Grant No. 70NANB8H4000, andthe United States government may have certain rights in the invention.

FIELD OF THE INVENTION

The invention is in the field of microfluidics and microarraytechnology. Apparatus, methods and integrated systems for detectingmolecular interactions are provided. The apparatus include microscalearrays which are movable or fixed in a microfluidic system. The systemsare capable of performing integrated manipulation and analysis in avariety of biological, biochemical and chemical experiments, including,e.g., DNA sequencing. Applications to chemical and biological systems,e.g., nucleic acid sequencing, enzyme kinetics, diagnostics, compoundscreening, and the like are provided.

BACKGROUND OF THE INVENTION

The development of microfluidic technologies by the inventors and theirco-workers has provided a fundamental paradigm shift in how artificialbiological and chemical processes are performed. In particular, theinventors and their co-workers have provided microfluidic systems whichdramatically increase throughput for biological and chemical methods, aswell as greatly reducing reagent costs for the methods. In thesemicrofluidic systems, small volumes of fluid (e.g., on the order of afew nanoliters to a few microliters) are moved through microchannels(e.g., in glass or polymer microfluidic devices) by electrokinetic orpressure-based mechanisms. Fluids can be mixed, and the results of themixing experiments determined by monitoring a detectable signal fromproducts of the mixing experiments.

Complete integrated systems with fluid handling, signal detection,sample storage and sample accessing are available. For example, Parce etal. “High Throughput Screening Assay Systems in Microscale FluidicDevices” WO 98/00231 and Knapp et al. “Closed Loop BiochemicalAnalyzers” (WO 98/45481; PCT/US98/06723) provide pioneering technologyfor the integration of microfluidics and sample selection andmanipulation. For example, in WO 98/45481, microfluidic apparatus,methods and integrated systems are provided for performing a largenumber of iterative, successive, or parallel fluid manipulations. Forexample, integrated sequencing systems, apparatus and methods areprovided for sequencing nucleic acids (as well as for many other fluidicoperations, e.g., those benefiting from automation of iterative fluidmanipulation). This ability to iteratively sequence a large nucleic acid(or a large number of nucleic acids) provides for increased rates ofsequencing, as well as lower sequencing reagent costs. Applications tocompound screening, enzyme kinetic determination, nucleic acidhybridization kinetics and many other processes are also described byKnapp et al.

As an alternative to microfluidic approaches, small scale array basedtechnologies can also increase throughput of screening, sequencing, andother chemical and biological methods, providing robust chemistries fora variety of screening, sequencing and other applications. Fixedsolid-phase arrays of nucleic acids, proteins, and other chemicals havebeen developed by a number of investigators. For example, U.S. Pat. No.5,202,231, to Drmanac et al. and, e.g., in Drmanac et al. (1989)Genomics 4:114-128 describe sequencing by hybridization to arrays ofoligonucleotides. Many other applications of array-based technologiesare commercially available from e.g., Affymetrix, Inc. (Santa Clara,Calif.), Hyseq Technologies, Inc. (Sunnyvale, Calif.) and others.Example applications of array technologies are described e.g., in Fodor(1997) “Genes, Chips and the Human Genome” FASEB Journal. 11:121-121;Fodor (1997) “Massively Parallel Genomics” Science. 277:393-395; Chee etal. (1996) “Accessing Genetic Information with High-Density DNA Arrays”Science 274:610-614; and Drmanac et al. (1998) “Accurate sequencing byhybridization for DNA diagnostics and individual genomics.” NatureBiotechnology 16: 54-58.

The present invention is a pioneering invention in the field ofmicrofluidics and mobile array technologies, coupling the fluid handlingcapabilities of microfluidic systems with the robust chemistriesavailable through array technologies (e.g., solid phase chemistries) tofacilitate laboratory and industrial processes. Many applications andvariations will be apparent upon complete review of this disclosure.

SUMMARY OF THE INVENTION

The present invention provides microfluidic arrays. The arrays includeparticle sets (or “packets”) which can be mobile or fixed in position,e.g., within a microfluidic system. The particle sets can include fixedchemical components or can be modifiable. The arrays are used in a widevariety of assays, as chemical synthesis machines, as nucleic acid orpolypeptide sequencing devices, as affinity purification devices, ascalibration and marker devices, as molecular capture devices, asmolecular switches and in a wide variety of other applications whichwill be apparent upon further review.

In one implementation, the invention provides microfluidic devicescomprising one or more array(s) of particles. The device includes a bodystructure having a microscale cavity (e.g., microchannel, microchannelnetwork, microwell, microreservoir or combination thereof) disposedwithin the body structure. Within the microscale cavity, an orderedarray of a plurality of sets of particles (each particle set isconstituted of similar or identical particle “members” or “types”)constitute the array. The array is optionally mobile (e.g., flowable ina microfluidic system, with flow being in either the same or in adifferent direction relative to fluid flow) or can be fixed (e.g.,having flowable reagents flowed across the system).

The arrays of the invention include a plurality of particle sets. Theprecise location of the particle sets within the arrays is not critical,and can take many configurations. In one simple embodiment, particlesets abut in channels. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100,1,000, 10,000, 100,000 or more particle sets can abut in a singlechannel. Alternatively, non-abutting sets of particles dispersed withina microfluidic device can also be used, e.g., where the spatial locationof each set of particles is known or can be determined. Fluidic reagentsand particles are optionally flowed through the same or throughdifferent microchannels (or other microfluidic structures such as wellsor chambers). For example, fluidic reagents are optionally flowed from afirst channel into a second channel which includes particle sets of thearray.

Particles (alternatively “microparticles”) of the arrays of theinvention can be essentially any discrete material which can be flowedthrough a microscale system. Example particles include beads andbiological cells. For example, polymer beads, silica beads, ceramicbeads, clay beads, glass beads, magnetic beads, metallic beads,inorganic beads, and organic beads can be used. The particles can haveessentially any shape, e.g., spherical, helical, irregular, spheroid,rod-shaped, cone-shaped, disk shaped, cubic, polyhedral or a combinationthereof. Particles are optionally coupled to reagents, affinity matrixmaterials, or the like, e.g., nucleic acid synthesis reagents, peptidesynthesis reagents, polymer synthesis reagents, nucleic acids,nucleotides, nucleobases, nucleosides, peptides, amino acids, monomers,cells, biological samples, synthetic molecules, or combinations thereof.Particles optionally serve many purposes within the arrays, includingacting as blank particles, dummy particles, calibration particles,sample particles, reagent particles, test particles, and molecularcapture particles, e.g., to capture a sample at low concentration.Additionally the particles are used to provide particle retentionelements. Particles are sized to pass through selected microchannelregions (or other microscale elements). Accordingly, particles willrange in size, depending on the application, e.g., from about 0.1 toabout 500 microns in at least one cross-sectional dimension.

In one aspect, the microfluidic system comprises an intersection of atleast two microchannels. At least one member of the particle array istransported within a first of the at least two channels to a pointproximal to or within the channel intersection. At least one of thereagents is transported through a second of the at least twointersecting microchannels to a point proximal to or within the channelintersection. The at least one member of the particle array and the atleast one reagent are contacted proximal to or within the channelintersection.

Methods of sequencing nucleic acids are provided. In the methods, afirst set of particles comprising at least one set of nucleic acidtemplates is provided, e.g., in a first microfluidic channel. A train ofreagents (i.e., an ordered or semi-ordered arrangement of fluidicreagents in a channel) comprising a plurality of sequencing reagents isflowed across the first set of particles, or the first set of particlesis flowed through the reagent train, depending on the application. Thisresults in contacting the at least one set of nucleic acid templateswith the plurality of sequencing reagents. Signals resulting fromexposure of the first set of particles to the reagent train areselected, thereby providing a portion of sequence of the nucleic acidtemplate. For example, the reagent train can include a polymerase, asufurylase, an apyrase, an inorganic phosphate, ATP, a thermostablepolymerase, luciferin, luciferase, an endonuclease, an exonuclease,Mg⁺⁺, a molecular crowding agent, a buffer, a dNTP, a dNTP analog, afluorescent nucleotide, a chain terminating nucleotide, a reversiblechain terminating nucleotide, a phosphatase, a reducing agent, anintercalator, a salt, DTT, BSA, a detergent (e.g., Triton® or Tween®),chemicals to inhibit or enhance EO flow (e.g., polyacrylamide), or othersequencing reagent. One preferred use for the arrays of the invention issequencing by “synthesis” or “incorporation,” e.g., pyrosequencing. Forexample, the reagent train or array optionally include reagents forsequencing nucleic acid templates by pyrosequencing. A variety of othersequencing approaches are described herein.

Steps in the methods herein can be performed repeatedly or reiterativelyfor chemistries such as sequencing that involve repetitive synthesisand/or analysis steps. As reagents are depleted e.g., in the reagenttrain noted above, the method further optionally includes flowing asecond train of reagents comprising a plurality of sequencing reagentsacross the first set of particles, or flowing the first set of particlesthrough the second reagent. Alternatively, reagents are flowed in excessfor a period of time, after which the channel(s) are rinsed, e.g., witha buffer before flowing a second reagent.

To further avoid contamination between repetitions or steps, methods areprovided for loading and unloading reagents from a microfluidic deviceusing a pair of split reagent wells and a pair of split waste wells.

Integrated systems and methods for performing fluidic analysis of samplematerials in a microfluidic system having a particle array are alsoprovided. For example, an integrated microfluidic system is providedwhich has a microfluidic device with the particle array, a materialtransport system, and a fluidic interface in fluid communication withthe particle array. The interface samples a plurality of materials fromone or a plurality of sources of materials and introduces the materialsinto contact with the particle array.

In the integrated methods, a first material from the plurality ofmaterials is sampled with the fluidic interface. The first material isintroduced into contact with at least one member of the particle array,whereupon the first sample material and at least a first member of theparticle array react. A reaction product of the first sample materialand the particle array is then analyzed and a second material (which maybe the same as or different than the first material) is selected, basedupon the reaction product. The second material is contacted with theparticle array, where the second material and at least a second memberof the particle array react. A second reaction product of the secondmaterial and the particle array is analyzed, thereby providing a fluidicanalysis of the first and second materials.

For example, in sequencing applications, the first material can includea first DNA sequencing template, a first sequencing primer, or a firstsequencing reagent while the second material can include a second DNAtemplate, a second sequencing primer, or a second sequencing reagent.The array, in this example, includes a first mixture of reagents havingDNA sequencing reagents or DNA templates. The first reaction productincludes products of a DNA sequencing reaction (e.g., primer extension,sequencing by incorporation, e.g., by the pyrosequencing reaction or thelike). A second sequencing primer is selected for inclusion in thesecond mixture of reagents based upon the products of the DNA sequencingreaction. Optionally, a third material is selected based upon theresults of the analysis of the second reaction product. The thirdmaterial is optionally introduced into proximity with the array,whereupon the third material and the array react. As above, the thirdreaction product is analyzed. This process is optionally reiteratedseveral times (e.g., easily 10 times or more, often 100-1,000 times ormore). Indeed, the process can be repeated thousands of times in asingle experiment, e.g., to sequence a long stretch of DNA, or the like.Ordinarily, the integrated system includes a computer for performing orassisting in selection of the second material. The integrated system canalso include fluid handling elements, e.g., electrokinetic or pressureflow controllers.

Systems for optimizing or performing a desired chemical reaction areprovided. The system includes a microfluidic device which includes amicroscale cavity having a particle array disposed therein. The particlearray includes a plurality of particle sets. The system includes anelectrokinetic or pressure based fluid direction system for transportinga selected volume of a first reactant to the array, or for reconfiguringthe position of the array or for reconfiguring the arrangement of arraymembers, or for loading array members (e.g., constituting a plurality ofarray sets) into the microscale cavity. The system also includes acontrol system, e.g., including a computer, which instructs the fluiddirection system to deliver a first selected volume of first reactant tothe array, or for moving members of the array into proximity with thefirst reactant, where contacting or mixing of the first reactant and atleast one member of the array produces a first chemical reactionproduct. The control system optionally directs a plurality of mixings ofthe first reactant and the array (e.g., by electrokinetic and/orpressure based manipulation of reagent or array member as describedherein), wherein a reaction condition selected from: temperature, pH,and time is systematically varied in separate mixings reactions. Thesystem typically includes a detection system for detecting the firstchemical reaction product e.g., as set forth above and supra. Otheroptional elements include a temperature control element for controllingtemperature of reaction of the first and second element, a source ofacid, a source of base and a source of reactants, reagents, arraymembers, or the like.

In one aspect, the system instructs the fluid direction system tocontact a second selected volume of the first or a second reactant withthe array. This contact produces a second chemical reaction product.

The particles of the arrays optionally include a tag and one or more ofthe particle modification reagents comprising an anti-tag ligand. A“tag” is a component that can be detected, directly or indirectly (e.g.,by binding to a detectable element). Exemplar tag and anti-tag ligandsinclude nucleic acids; nucleic acid binding molecules, amidin, biotin,avidin, streptavidin, antibodies, antibody ligands, carbohydratemolecules, carbohydrate molecule binding reagents, proteins, proteinbinding molecules, organic molecules, organic molecule binding reagents,receptors, receptor ligands, etc. The particle modification reagent canalso include a functional domain, e.g., independently selected fromthose noted for the tag and tag ligand. For example, in one embodiment,the one or more particle modification reagent has a nucleic acid havinga biotin or avidin attached thereto.

Wash buffers, heat application, or an electric pulse are optionally usedto strip components from arrays, thereby changing the array members. Newcomponents can be added to the array members following such washing. Forexample one or more particle modification reagents can be removed fromone or more of the particle sets following washing, to provide one ormore stripped particle sets. At least one additional particlemodification reagent can be flowed across the one or more strippedparticle set, thereby producing an additional particle set.

Many additional aspects of the invention will be apparent upon reviewincluding uses of the devices and systems of the invention, methods ofmanufacture of the devices and systems of the invention, kits forpracticing the methods of the invention and the like. For example, kitscomprising any of the devices or systems set forth above, or elementsthereof, in conjunction with packaging materials (e.g., containers,sealable plastic bags etc.) and instructions for using the devices,e.g., to practice the methods herein, are also contemplated. Methods ofManufacture and manufactured devices comprising arrays or array membersare set forth in detail herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1, panels A and B, is a schematic top view and expanded view of anexample microfluidic system comprising array elements.

FIG. 2, panels A and B provide side view schematics of a microchanneland particle retention region arrangement.

FIG. 3, panels A and B, is a top view and expanded view schematic of anexample microfluidic system comprising array elements.

FIG. 4, panels A, B, and C provide top view and expanded view schematicsof an example microfluidic system comprising array elements.

FIG. 5 is a side-view schematic of a main channel with reagentintroduction channels for sequencing nucleic acids.

FIG. 6 is a schematic of a sipper for reagent/array train introductioninto microfluidic devices.

FIG. 7 is a schematic of a multiplexing arrangement useful for DNAsequencing.

FIG. 8 is a schematic showing a device with pipettor access to aparticle library.

FIG. 9, panels A and B, provide a schematic of a device with particlepackets in channels having reagent flow across the top of the particles.Panel A is a cross section. Panel B is a top view.

FIG. 10 is a schematic illustration of a top view of a device showingloading of bead packets.

FIG. 11, panels A, B and C, show schematic illustrations of alternatebead packet loading embodiments. Panel A is a top view. Panel B is a topview. Panel C is a top view.

FIG. 12 is a schematic of a system comprising a computer, detector,array, sampling system, and particle library.

FIG. 13 is a schematic of a reversible terminator labeling strategy.

FIG. 14 is a side view of a capillary or microchannel containing aporous barrier formed from a plurality of particles which captures orretains a solid phase reaction element, e.g., another set of particles.

FIG. 15 is a side view of a capillary or microchannel in a housing,which housing comprises a porous barrier.

FIG. 16 is a side view of a capillary or microchannel comprising anintegral or formed porous barrier made from a set of particles. Theporous barrier is used, e.g., to capture multiple packets, i.e., sets ofparticles.

FIG. 17 is a schematic illustrating sequencing by synthesis in a highthroughput system.

FIG. 18, panels A, B, and C, provides schematic views of examplemicrofluidic devices useful in split well loading and unloading, e.g.,of reagents and particles, e.g., to avoid contamination.

DEFINITIONS

Unless indicated to the contrary, the following definitions supplementthose in the art.

“Microfluidic,” as used herein, refers to a system or device havingfluidic conduits or chambers that are generally fabricated at the micronto submicron scale, e.g., typically having at least one cross-sectionaldimension in the range of from about 0.1 μm to about 500 μm. Themicrofluidic system of the invention is fabricated from materials thatare compatible with the conditions present in the particular experimentof interest. Such conditions include, but are not limited to, pH,temperature, ionic concentration, pressure, and application ofelectrical fields. The materials of the device are also chosen for theirinertness to components of the experiment to be carried out in thedevice. Such materials include, but are not limited to, glass, quartz,silicon, and polymeric substrates, e.g., plastics, depending on theintended application.

A “microscale cavity” is a conduit or chamber having at least onedimension between about 0.1 and 500 microns.

A “microchannel” is a channel having at least one microscale dimension,as noted above. A microchannel optionally connects one or moreadditional structure for moving or containing fluidic or semi-fluidic(e.g., gel- or polymer solution-entrapped) components.

An “ordered array of a plurality of sets of particles” is an array ofparticle sets (each particle set is constituted of similar or identicalparticle “members” or “types”) having a spatial arrangement. The spatialarrangement of particle sets can be selected or random. In a preferredembodiment, the spatial arrangement is selected. The arrangement can beknown or unknown. In a preferred embodiment, the spatial arrangement ofparticle sets is known.

A “set” of particles is a group or “packet” of particles having similaror identical constituents.

A “particle movement region” is a region of a microscale element inwhich particles are moved. A “fluid movement region” is a region of amicroscale element in which fluidic components are moved. As discussedsupra, fluidic and particulate elements are moved by any of a variety offorces, including capillary, pressure, electrokinetic and the like.

A “particle retention region” is a region of a microscale element inwhich particles can be localized, e.g., by placing a physical barrier orporous matrix within or proximal to the retention region, by applicationof magnetic or electric fields, by application of pressure, or the like.For example, a porous matrix optionally comprises a fixed set ofparticles, e.g., particles about 100 μm to about 200 μm incross-sectional dimension, within a microchannel.

A “microwell plate” is a substrate comprising a plurality of regionswhich retain one or more fluidic components.

A “pipettor channel” is a channel in which components can be moved froma source to a microscale element such as a second channel or reservoir.The source can be internal or external to a microfluidic devicecomprising the pipettor channel.

Two components are “physically associated” when they are in direct orindirect contact.

A particle array is “mobile” when a plurality of sets of the array canbe moved in a selected or selectable manner, e.g., by electrokinetic,pressure-based or capillary fluid movement systems, or a combination ofmovement systems.

A “serial” stream of components, e.g., reagents is a train of reagents,is an ordered linear arrangement of the components.

A “parallel” set of components is an array of components which are in aplurality of linear arrangements, e.g., separate linear arrangements, ofthe components.

A “particle modification reagent” is a reagent that binds to orchemically alters one or more component that is physically associatedwith the particle.

DETAILED DESCRIPTION

The present invention provides microfluidic arrays. The array componentscan be mobile or fixed. They can also be of a selected type or typeswitchable, and can incorporate any of a wide variety of chemical orbiochemical components. The arrays are broadly useful as tools forscreening assay components, biopolymer sequencing, drug screening, assaynormalization, as miniaturized chemical and biochemical synthesismachines, as molecular switches, as fluidic logic circuits and a varietyof other applications that will become apparent upon complete review ofthis disclosure. The arrays can be components of integrated systems.

Methods of performing a plurality of chemical reactions in a microscaledevice are an aspect of the invention. In the methods, an array within amicrofluidic system (e.g., having a body structure with a microscaleinterior cavity, etc. as described above) is provided. One or moreliquid reagent is flowed into the interior cavity and into contact withparticle sets of the array. The liquid reagent chemically reacts withone or more of the plurality of particles, thereby providing a chemicalreaction in the microscale device.

Optionally, one or more of the plurality of sets of particles of thearray (or the entire array or a substantial portion of) is moved into orthrough an intersection of at least two channels present in amicrofluidic system. Mixing can occur in the intersections of channels,or within chambers, channels, wells, reservoirs, or the like. Thus, inthe methods of the invention, at least one of the plurality of sets ofparticles can be moved through at least one of the at least two channelsinto an intersection of the at least two channels, while (separately orsimultaneously) flowing the liquid reagent through a second of the atleast two channels into the channel intersection, where the liquidreagent flows into contact with at least one set of particles of thearray. In one aspect, the cavity comprises a main channel having aplurality of intersecting side channels, forming a plurality of channelintersections between the main channel and each of the intersecting sidechannels. The methods optionally include transporting at least one ofthe plurality of sets of particles in the main channel into at least twoof the plurality of channel intersections.

Similarly, in one aspect, the method includes transporting at least onefluidic reagent through at least one of the side channels into at leastone of the plurality of channel intersections, where the reagent flowsinto at least one of the plurality of sets of particles in the mainchannel. Alternatively, one or more of the particle sets of the array isflowed through a single microchannel and various reagents, e.g., liquidreagents, are optionally flowed through the particles or the particlesare flowed through the reagent. The liquid reagent is optionally flowedthrough a capillary fluidly coupled to the single microchannel, e.g., acapillary that sips fluid from a microwell plate into a microfluidicdevice, e.g., a single channel or multi-channel device.

Methods also optionally include moving particles (or reagents) into theinterior cavity. For example, in one embodiment, the interior cavity hasa broad channel with narrow channels within the broad channel. Thenarrow channels are deeper in at least one dimension than the broadchannel. A plurality of sets of particles are transported into one ormore of the narrow channels to form the array. Optionally, a liquidreagent is also (subsequently or previously) transported through thebroad channel and the narrow channel and into contact with the pluralityof sets of particles. For example, a liquid reagent is optionally sippedfrom a microtiter plate by a capillary fluidly coupled to a microfluidicdevice, in which device the liquid reagent contacts one or more particleset. Microparticles are also optionally brought into a microfluidicdevice through a sipper capillary attached to a microfluidic device.Exemplar liquid reagents include those described above such as nucleicacid sequencing reagents.

One preferred use for the arrays of the invention is nucleic acid orprotein sequencing (or sequencing monomer elements of any polymer). Forexample the methods of the invention optionally include sequencing byhybridization, sequencing by synthesis or incorporation, sequencing byphotobleaching, sequencing by intercalation, specific detection ofnucleic acid polymorphisms, specific detection of a nucleic acid,diagnosing or predicting prognosis of a disease or infectious conditionassociated with presence or absence of a nucleic acid in a biologicalsample, diagnosing or predicting prognosis of a disease or infectiouscondition associated with presence or absence of a protein in abiological sample, serial or parallel hybridization between multipleliquid reagents and members of the array, and serial or paralleldetection of results of multiple hybridization reactions between liquidreagents and members of the array.

Methods of contacting samples and reagents in a microfluidic system arealso provided. In these methods, a plurality of members of a particlearray which includes a plurality of samples is transported to a selectedlocation within the microfluidic system. Simultaneously, separately orsequentially, the reagents are also transported within the microfluidicsystem such that members of the particle array and the reagents arecontacted. Members of the particle array and the reagents are optionallyrepeatedly transported sequentially or simultaneously within themicrofluidic system. In microfluidic systems having one or more emissiondetectors, the method optionally includes transporting the plurality ofparticle members past the one or more emission detectors, before,during, or after contacting the plurality of particle members with oneor more of the reagents.

Array Configurations

As noted, the devices and systems of the invention typically include abody structure having a microscale cavity (e.g., microchannel,microchannel network, microwell, microreservoir or combination thereof)disposed within the body structure. Within the microscale cavity, anordered array of a plurality of sets of particles constitute the array.The array is optionally mobile (e.g., flowable in a microfluidic system)or can be fixed (e.g., having flowable reagents flowed across thesystem). A fixed array of particles optionally comprises a porousbarrier to capture or retain other solid phase components, e.g., anotherset of particles.

Mobile Arrays

The arrays of the invention are optionally mobile. Arrays compriseparticle sets such as beads, microspheres, cells, fluidly transportablesubstrates, or the like. The particles are optionally moved by flowingfluids comprising the particles through or to a desired location, e.g.,within a microfluidic system. Particles or reagents can be moved withinmicrofluidic arrays by any of a variety of techniques, including fluidicpressure, atmospheric pressure, capillary force, gravity,electrokinesis, electric and magnetic fields, and centripetal orcentrifugal force.

The inventors and their co-workers have provided a variety ofmicrofluidic systems in which the arrays of the invention can beconstructed. For example, Ramsey WO 96/04547 provides a variety ofmicrofluidic systems. See also, Ramsey et al. (1995), Nature Med.1(10):1093-1096; Kopf-Sill et al. (1997) “Complexity and performance ofon-chip biochemical assays,” SPIE 2978:172-179 February 10-11; Bousse etal. (1998) “Parallelism in integrated fluidic circuits,” SPIE3259:179-186; Chow et al. U.S. Pat. No. 5,800,690; Kopf-Sill et al. U.S.Pat. No. 5,842,787; Parce et al., U.S. Pat. No. 5,779,868; Parce, U.S.Pat. No. 5,699,157; U.S. Pat. No. 5,852,495 (J. Wallace Parce) issuedDec. 22, 1998; U.S. Pat. No. 5,869,004 (J. Wallace Parce et al.) issuedFeb. 9, 1999, U.S. Pat. No. 5,876,675 (Colin B. Kennedy) issued Mar. 2,1999; U.S. Pat. No. 5,880,071 (J. Wallace Parce et al.) issued Mar. 9,1999; U.S. Pat. No. 5,882,465 (Richard J. McReynolds) issued Mar. 16,1999; U.S. Pat. No. 5,885,470 (J. Wallace Parce et al.) issued Mar. 23,1999; U.S. Pat. No. 5,942,443 (J. Wallace Parce et al.) issued Aug. 24,1999; U.S. Pat. No. 5,948,227 (Robert S. Dubrow) issued Sep. 7, 1999;U.S. Pat. No. 5,955,028 (Calvin Y. H. Chow) issued Sep. 21, 1999; U.S.Pat. No. 5,957,579 (Anne R. Kopf-Sill et al.) issued Sep. 28, 1999; U.S.Pat. No. 5,958,203 (J. Wallace Parce et al.) issued Sep. 28, 1999; U.S.Pat. No. 5,958,694 (Theo T. Nikiforov) issued Sep. 28, 1999; and U.S.Pat. No. 5,959,291 (Morten J. Jensen) issued Sep. 28, 1999; Parce et al.WO 98/00231; Parce et al. WO 98/00705; Chow et al. WO 98/00707; Parce etal. WO 98/02728; Chow WO 98/05424; Parce WO 98/22811; Knapp et al., WO98/45481; Nikiforov et al. WO 98/45929; Parce et al. WO 98/46438; Dubrowet al., WO 98/49548; Manz, WO 98/55852; WO 98/56505; WO 98/56956; WO99/00649; WO 99/10735; WO 99/12016; WO 99/16162; WO 99/19056; WO99/19516; WO 99/29497; WO 99/31495; WO 99/34205; WO 99/43432; and WO99/44217; U.S. Pat. No. 5,296,114; and e.g., EP 0 620 432 A1; Seiler etal. (1994) Mitt Gebiete Lebensm. Hyg. 85:59-68; Seiler et al. (1994)Anal. Chem. 66:3485-3491; Jacobson et al. (1994) “Effects of InjectionSchemes and Column Geometry on the Performance of MicrochipElectrophoresis Devices” Anal. Chem. 66: 66. 1107-1113; Jacobsen et al.(1994) “Open Channel Electrochromatography on a Microchip” Anal. Chem.66:2369-2373; Jacobsen et al. (1994) “Precolumn Reactions withElectrophoretic Analysis Integrated on Microchip” Anal. Chem.66:4127-4132; Jacobsen et al. (1994) “Effects of Injection Schemes andColumn Geometry on the Performance of Microchip ElectrophoresisDevices.” Anal. Chem. 66:1107-1113; Jacobsen et al. (1994) “High SpeedSeparations on a Microchip.” Anal. Chem. 66:1114-1118; Jacobsen andRamsey (1995) “Microchip electrophoresis with sample stacking”Electrophoresis 16:481-486; Jacobsen et al. (1995) “Fused QuartzSubstrates for Microchip Electrophoresis” Anal. Chem. 67: 2059-2063;Harrison et al. (1992) “Capillary Electrophoresis and Sample InjectionSystems Integrated on a Planar Glass Chip.” Anal. Chem. 64:1926-1932;Harrison et al. (1993) “Micromachining a Miniaturized CapillaryElectrophoresis-Based Chemical Analysis System on a Chip.” Science 261:895-897; Harrison and Glavania (1993) “Towards MiniaturizedElectrophoresis and Chemical System Analysis Systems on Silicon: AnAlternative to Chemical Sensors.” Sensors and Actuators 10:107-116; Fanand Harrison (1994) “Micromachining of Capillary ElectrophoresisInjectors and Separators on Glass Chips and Evaluation of Flow atCapillary Intersections. Anal. Chem. 66: 177-184; Effenhauser et al.(1993) “Glass Chips for High-Speed Capillary Electrophoresis Separationswith Submicrometer Plate Heights” Anal. Chem. 65:2637-2642; Effenhauseret al. (1994) “High-Speed Separation of Antisense Oligonucleotides on aMicromachined Capillary Electrophoresis Device.” Anal. Chem.66:2949-2953; and Kovacs EP 0376611 A2.

In general, these microfluidic systems can be adapted for use in thepresent invention, i.e., by introducing arrays into the microfluidicsystems, e.g., for the assays set forth herein. A variety of arraycomponent introduction approaches for introducing array elements intomicrofluidic systems are noted herein.

Movement of Particles within Microfluidic Systems

The microfluidic devices which include arrays also can include otherfeatures of microscale systems, such as fluid transport systems whichdirect particle movement within the array channel, incorporating anymovement mechanism set forth herein (e.g., fluid pressure sources formodulating fluid pressure in the array channel, electrokineticcontrollers for modulating voltage or current in the array channel,gravity flow modulators, magnetic control elements for modulating amagnetic field within the array channel, or combinations thereof). Themicroscale cavity can also include fluid manipulation elements such as aparallel stream fluidic converter, i.e., a converter which facilitatesconversion of at least one serial stream of reagents into parallelstreams of reagents for parallel delivery of reagents to a reaction sitewithin the microscale cavity. For example, a capillary is optionallyused to sip a sample or samples from a microtiter plate and to deliverit to one of a plurality of channels, e.g., parallel reaction or assaychannels. In addition, particle sets are optionally loaded into one ormore channels of a microfluidic device through one sipper fluidlycoupled to each of the one or more channels and to a sample or particlesource, such as a microwell plate. Indeed, one advantage of the presentsystem is the ability to provide parallel streams of reagents to samplesfixed on arrays. This is particularly advantageous, because thevolumetric accuracy requirements for delivery of reagents is often lessthan the volumetric requirements for samples. A wide variety ofintegrated microfluidic systems comprising serial to parallel fluidmanipulation are described in Bousse et al., “Parallelism in integratedfluidic circuits,” SPIE 3259:179-186 (1998) and in CLOSED LOOPBIOCHEMICAL ANALYZERS; WO 98/45481 and the references therein.

Electrokinetic Controllers

One method of achieving transport or movement of particles throughmicrofluidic channels is by electrokinetic material transport.“Electrokinetic material transport systems,” as used herein, includessystems that transport and direct materials within a microchannel and/orchamber containing structure, through the application of electricalfields to the materials, thereby causing material movement through andamong the channel and/or chambers, i.e., cations will move toward anegative electrode, while anions will move toward a positive electrode.For example, movement of fluids toward or away from a cathode or anodecan cause movement of particles suspended within the fluid (or evenparticles over which the fluid flows). Similarly, the particles can becharged, in which case they will move toward an oppositely chargedelectrode (indeed, in this case, it is possible to achieve fluid flow inone direction while achieving particle flow in the opposite direction).In this embodiment, the fluid can be immobile or flowing.

In general, electrokinetic material transport and direction systemsinclude those systems that rely upon the electrophoretic mobility ofcharged species within the electric field applied to the structure. Suchsystems are more particularly referred to as electrophoretic materialtransport systems. For electrophoretic applications, the walls ofinterior channels of the electrokinetic transport system are optionallycharged or uncharged. Typical electrokinetic transport systems are madeof glass, charged polymers, and uncharged polymers. The interiorchannels are optionally coated with a material which alters the surfacecharge of the channel.

A variety of electrokinetic controllers are described, e.g., in RamseyWO 96/04547, Parce et al. WO 98/46438 and Dubrow et al., WO 98/49548, aswell as a variety of other references noted herein.

To provide appropriate electric fields, the system generally includes avoltage controller that is capable of applying selectable voltagelevels, simultaneously, to each of the reservoirs, including the ground.Such a voltage controller is optionally implemented using multiplevoltage dividers and multiple relays to obtain the selectable voltagelevels. Alternatively, multiple independent voltage sources are used.The voltage controller is electrically connected to each of thereservoirs via an electrode positioned or fabricated within each of theplurality of reservoirs. In one embodiment, multiple electrodes arepositioned to provide for switching of the electric field direction in amicrochannel, thereby causing the analytes to travel a longer distancethan the physical length of the microchannel. Use of electrokinetictransport to control material movement in interconnected channelstructures was described, e.g., in WO 96/04547 to Ramsey. An exemplarycontroller is described in U.S. Pat. No. 5,800,690. Modulating voltagesare concomitantly applied to the various reservoirs to affect a desiredfluid flow characteristic, e.g., continuous or discontinuous (e.g., aregularly pulsed field causing the sample to oscillate direction oftravel) flow of labeled components toward a waste reservoir.Particularly, modulation of the voltages applied at the variousreservoirs can move and direct fluid flow through the interconnectedchannel structure of the device.

Other Particle and Fluid Movement Approaches

Other methods of transport are also available for situations in whichelectrokinetic methods are not desirable. For example, sampleintroduction and reaction are optionally carried out in a pressure-basedsystem to avoid electrokinetic biasing during sample mixing and highthroughput systems typically use pressure induced sample introduction.Pressure based flow is also desirable in systems in which electrokinetictransport is also used. For example, pressure based flow is optionallyused for introducing and reacting reagents in a system in which theproducts are electrophoretically separated. In the present inventionparticle arrays are optionally loaded using electrokinetic fluid controland other reagents are flowed through the particle arrays underpressure.

Pressure is optionally applied to microscale elements, e.g., to achannel, region, or reservoir, to achieve fluid movement using any of avariety of techniques. Fluid flow and flow of materials suspended orsolubilized within the fluid, including cells or particle sets, isoptionally regulated by pressure based mechanisms such as those basedupon fluid displacement, e.g., using a piston, pressure diaphragm,vacuum pump, probe or the like to displace liquid and raise or lower thepressure at a site in the microfluidic system. The pressure isoptionally pneumatic, e.g., a pressurized gas, or uses hydraulic forces,e.g., pressurized liquid, or alternatively, uses a positive displacementmechanism, i.e., a plunger fitted into a material reservoir, for forcingmaterial through a channel or other conduit, or is a combination of suchforces.

In some embodiments, a vacuum source is applied to a reservoir or wellat one end of a channel to draw a fluidic material through the channel.For example, a vacuum source is optionally placed at a reservoir in thepresent devices for drawing fluid into or through a channel, e.g.,through a porous particle retention element, e.g., a particle set.Pressure or vacuum sources are optionally supplied external to thedevice or system, e.g., external vacuum or pressure pumps sealablyfitted to the inlet or outlet of the channel, or they are internal tothe device, e.g., microfabricated pumps integrated into the device andoperably linked to the channel. Examples of microfabricated pumps havebeen widely described in the art. See, e.g., published InternationalApplication No. WO 97/02357.

Hydrostatic, wicking and capillary forces are also optionally used toprovide fluid pressure for continuous fluid flow of materials such asenzymes, substrates, modulators, or protein mixtures. See, e.g., “METHODAND APPARATUS FOR CONTINUOUS LIQUID FLOW IN MICROSCALE CHANNELS USINGPRESSURE INJECTION, WICKING AND ELECTROKINETIC INJECTION,” by Alajoki etal. in U.S. Pat. No. 6,416,642. In these methods, an adsorbent materialor branched capillary structure is placed in fluidic contact with aregion where pressure is applied, thereby causing fluid to move towardsthe adsorbent material or branched capillary structure. The capillaryforces are optionally used in conjunction with the electrokinetic orpressure-based flow in the present invention. The capillary action pullsmaterial through a channel. For example a wick is optionally added todraw fluid through a porous matrix fixed in a microscale channel orcapillary.

Mechanisms for reducing adsorption of materials during fluid-based floware described in “PREVENTION OF SURFACE ADSORPTION IN MICROCHANNELS BYAPPLICATION OF ELECTRIC CURRENT DURING PRESSURE-INDUCED FLOW” filed May11, 1999 by Parce et al., application Ser. No. 09/310,027 (now U.S. Pat.No. 6,458,259). In brief, adsorption of components, proteins, enzymes,markers and other materials to channel walls or other microscalecomponents during pressure-based flow can be reduced by applying anelectric field such as an alternating current to the material duringflow. Alternatively, flow rate changes due to adsorption are detectedand the flow rate is adjusted by a change in pressure or voltage.

Mechanisms for focusing labeling reagents, enzymes, modulators, andother components into the center of microscale flow paths, which isuseful in increasing assay throughput by regularizing flow velocity,e.g., in pressure based flow, is described in “FOCUSING OFMICROPARTICLES IN MICROFLUIDIC SYSTEMS” by H. Garrett Wada et al., filedMay 17, 1999, application 60/134,472. In brief, sample materials arefocused into the center of a channel by forcing fluid flow from opposingside channels into the main channel comprising the cells, or by otherfluid manipulation.

In an alternate embodiment, microfluidic systems can be incorporatedinto centrifuge rotor devices, which are spun in a centrifuge. Fluidsand particles travel through the device due to gravitational andcentripetal/centrifugal pressure forces.

Fluid flow or particle flow in the present devices and methods isoptionally achieved using any one of the above techniques, alone or incombination.

Particle Retention Regions and Other Specific Configurations

As noted, the particle sets of the arrays of the invention can be fixedin place, or mobile. For example, the microscale cavity can have a firstmicrochannel which includes a particle movement region and a particleretention region. The particle retention region typically includes atleast one set of the plurality of sets of particles disposed within theparticle retention region, although it can include more than one set.The particle “retention” area or region optionally includes a region ofincreased or decreased microchannel depth or width or other physicalbarrier (groove, mesh, net, matrix, etc.), an electromagnetic field, aporous matrix (e.g., sieving matrices are optional components ofmicrofluidic systems, as described below), or other means of inhibitingparticle movement in or adjacent to the region.

For example, in embodiments where the microfluidic cavity comprising thearray has a first microchannel, the microchannel is optionallyconfigured to include at least one reagent flow region and at least oneparticle capture region. The at least one particle capture regionoptionally has an increased or decreased depth or width relative to theat least one reagent flow region, or is bounded by a region of increasedor decreased depth or width. For example, in one embodiment, the devicesinclude regions of width (or depth) sufficient to permit passage ofparticles, where the particle capture region is insufficient indimension to permit free passage of the particles. For example, whereparticles larger than 3-4 microns are used, a channel region having across sectional dimension smaller than 3-4 microns blocks flow of theparticles, while still permitting flow of fluids. Preferred particlesize ranges from about 0.1 microns to about 50 microns; accordingly, inthis embodiment, a physical barrier which does not permit passage of aselected particle within this range of particle sizes can be used as aparticle capture element. In one aspect, a “particle capture region” isbounded on at least one side by such particle capture elements. Inanother example, the reagent flow region comprises a cross-sectionaldimension comprising passage of selected particles and the particleretention or capture region abuts a narrow channel region comprising adimension sufficiently small to inhibit movement of a selected particlethrough the narrow channel. Such narrow channel regions are typicallyless than about 10 μm, more typically less than 5 μm. Preferable narrowchannel dimensions are about 5 μm or smaller or about 3 μm or smaller.

In other embodiments, the particle capture region, e.g., in a capillaryor microchannel, comprises a device to capture a set of particles, e.g.,chemically coated microspheres or other chemically coated solid phaseobjects, e.g., for a sequential chemistry assay. The device typicallycomprises a physical barrier, e.g., a porous barrier with a defined poresize, that captures solid phase objects having a larger mean diameterthan the pore size. The upstream end of the capillary or microchannel istypically inserted in a reagent, e.g., a liquid reagent, and thedownstream end is typically coupled to a vacuum source or electrokineticcontroller which transports the reagents across the bed of the capturedsolid phase. The first reagent brought into the channel is typically thesolid phase in a suitable buffer and subsequent reagents followsequentially, e.g., sequencing regents. For example, DNA coatedmicrospheres are optionally captured in a particle retention area forsequencing by synthesis, wherein subsequent reagents comprise dNTPsbrought in one at a time, e.g., with sufficient rinsing betweennucleotides. Other applications include DNA probes and the like. Thecaptured solid phase is typically captured in the particle retentionarea, e.g., by a physical barrier. Such barriers are optionally formedby a fixed particle set, by a sintered glass frit, e.g., a preformedfrit that has been inserted into the channel or a frit that has beenfabricated integral to the capillary or channel. Alternative barriers,include a frit, e.g., a fixed set of particles, formed from epoxy coatedmicrospheres, e.g., providing an appropriate pore size. The fixed set ofparticles, e.g., epoxy coated microspheres, form a porous barrier whichcaptures other particle sets or packets. For example, a set of particlesis optionally flowed through a capillary, e.g., with glue or epoxy. Thecapillary is centrifuged to flow the glue or epoxy resin over theparticles. Excess glue is then removed, leaving a fixed particle matrixwhich is optionally used to capture or retain flowing or mobile beads,e.g., chemically coated beads. Other materials used to form the barrierinclude, but are not limited to, glass, plastic, and other materialscomprising an appropriate pore size. The barrier or frit is optionallycontained in a housing, which housing is optionally a capillary or amachined or molded plastic or glass unit.

The flow region can be in the same plane or transverse to the particlecapture region. In typical embodiments, the microfluidic devicecomprising the microscale cavity includes a plurality of microchannels,which are optionally intersecting or non intersecting and whichoptionally fluidly connect with reservoirs, wells or the like. Themicrofluidic device can include, or be coupled to (e.g., through achannel, microchannel, sipper capillary, pipettor, pressure pipettor,electropipettor, etc.), external sources of reagents or particles,permitting loading of the particles, or reagents which interact with theparticles. Thus, in one embodiment, the device comprises a loadingchannel region (or particle loading channel region) coupled to a sourceof at least a first selected particle type or reagent coupled to themicroscale cavity. For example, the source of particle type or reagentcan be a microwell plate external to the body structure having at leastone well with the selected particle type or reagent, a well disposed onthe surface of the body structure comprising the selected particle typeor reagent, a reservoir disposed within the body structure comprisingthe selected particle type or reagent; a container external to the bodystructure comprising at least one compartment comprising the selectedparticle type or reagent, or a solid phase structure comprising theselected particle type or reagent.

The particle loading channel region is optionally fluidly coupled to oneor more of: a pipettor channel with a port external to the bodystructure, an electropipettor channel with a port external to the bodystructure, a sipper capillary, e.g., external to the body structure,fluidly coupled to one or more microwell plates, a pipettor channel witha port internal to the body structure, an internal channel within thebody structure fluidly coupled to a well on the surface of the bodystructure, an internal channel within the body structure fluidly coupledto a well within the body structure, or the like.

The ordered array optionally includes a plurality of sample setsassociated with the plurality of particle sets, where each sample set isphysically associated with one or more of the plurality of particlesets. In addition, the ordered array optionally includes a plurality ofreagent sets associated with the plurality of particle sets, where eachreagent set is physically associated with one or more of the pluralityof particle sets. These reagent and particle sets are optionallyassociated with each other, typically such that each reagent set andeach particle set is physically associated with one or more of theplurality of particle sets. Manufacturing methods for making the arrays,microfluidic devices, integrated systems and the like are also provided.

In addition to the aspects set forth above, in one specific set ofmethods, a plurality of particle sets is flowed through a firstmicroscale channel having a particle flow region having a height towidth aspect ratio greater than 1.

In another set of manufacturing embodiments, a first particle set isflowed through the particle flow region and the first particle set isfixed in a first location in the channel, resulting in a first fixedparticle set, e.g., forming a particle retention area to capture orretain other sets of particles. A second particle set is flowed throughthe particle flow region to a region abutting the first fixed particleset, where the first fixed particle set blocks further flow of thesecond particle set in the direction of flow, resulting in a secondfixed particle set. In some embodiments, a third particle set throughthe particle flow region to a region abutting the second fixed particleset, wherein the second fixed particle set blocks further flow of thethird particle set in the direction of flow, resulting in a third fixedparticle set. In some embodiments, the first particle set is fixed in aparticle retention region, which inhibits movement of selected particlesfrom the particle retention region e.g., by a physical barrier tomovement of the particles that is proximal to or within the particleretention region, a magnetic field proximal to or within the particleretention region, a chemical particle capture moiety that is proximal toor within the particle retention region or the like.

In one aspect, the first particle set is fixed in a particle retentionregion, which abuts the particle flow region. The particle retentionregion has a decreased or increased height to width aspect ratio ascompared to the particle flow region, which decreased or increasedheight to width aspect ratio, in combination with the dimensions of theparticle flow region, creates a physical barrier at the point where thedimensions vary and inhibits movement of the first particle set from theparticle retention region. Alterations in the aspect ratio can also beused to provide regions of faster or slower particle flow, depending onthe shape of the particles. In another aspect, the particle retentionarea is formed by a set of particles, e.g., epoxy coated particles,which has been fixed in position in the channel, e.g., in a housing orby glue or epoxy. Alternatively, a sintered glass frit or other porousmatrix is placed in the channel or fabricated within the channel to forma barrier.

In another set of manufacturing embodiments, methods of making amicrofluidic particle array are provided. In the methods, a microfluidicdevice comprising a microscale cavity is provided. One or more particlesets are flowed into the microscale cavity. One or more particlemodification reagents are flowed into contact with the one or moreparticle sets, thereby producing a plurality of particle sets comprisingthe microfluidic particle array.

Further understanding of specific configurations is developed byconsideration of the following specific embodiments.

FIG. 1 provides microfluidic device 1 comprising reagent wells 10-30,additional wells 40-70 and waste well 80. The wells are fluidlyconnected to microchannel 90 comprising intersections 100 and 120.Additional wells 40-70 are fluidly coupled to intersections 100-130.Channel constriction 140 provides a bead capture area. As shown indetail, constriction 140 traps beads/particles 150 in particle retentionregion 160. In one example of this embodiment, the particles are betweenabout 10 and about 4 microns, while particle retention region 160 isabout 15 microns and constriction 140, e.g., a narrow channel region, isabout 3 microns in diameter. Alternatively, the particles are largerthan about 12 microns and particle retention region 160 is about 20microns to about 50 microns and constriction 140 is about 3 microns toabout 12 microns. For particles larger than about 15 microns,constriction region 140 is less than about 15 microns. Various particlesizes are used with various constriction measurements. Typically, theconstriction is smaller than the particles of interest. An additionalembodiment utilizing only one channel is provided in FIG. 14. Channel orcapillary 1405 is optionally coupled to a sipper capillary that drawsreagents, e.g., from a microwell plate, and through the capillary, e.g.,through a pressure or electrokinetic controller.

FIG. 2, Panel A, provides microfluidic device 201 comprising channel210. In a region (e.g., a bottom region) of channel 210 several particleretention regions 220-250 retain particle sets 260-290. In this simpleembodiment, particle sets 260-290 constitute an array, or a portion ofan array. Particle sets 260-290 optionally comprise particle memberswhich are of smaller dimension than channel 210. In this configuration,particle sets 260-290 are optionally magnetic and held in place byapplication of an appropriate magnetic field. In other embodiments,channel 210 is of sufficiently narrow dimension that members of particlesets 260-290 cannot exit retention regions 220-250. This embodiment isdepicted in FIG. 2B. Effective channel dimensions can be altered duringoperation by addition of a matrix or size-exclusion gel to the channel.

FIG. 3, panels A and B provide an additional aspect of the invention.The channels and particles used are typically smaller than the onesshown. An expanded view is used for ease of illustration. Microfluidicdevice 301 comprises wells 310-340. The wells are fluidly connectedthrough a network of channels comprising channels 350-390 andintersections 400-410. Channel region 415 comprises particle sets420-440. These particle sets can be immobile or mobile. For example, theparticles are optionally held in place by a particle retention element,e.g., a porous barrier. Particle sets 420-440 are optionally the samesize and/or shape or different sizes and/or shapes. In addition, eachparticle set optionally comprises a different number of particles.

FIG. 4, panels A, B and C show an additional embodiment (the channeldimensions are shown larger than typical channel dimensions for ease ofillustration). Microfluidic device 4001 comprises wells 4005-4025. Thewells are fluidly connected through channels 4030-4040 and throughdouble-depth channels 4045-4050 and single depth wide channel regions4060-4070. Double-depth channel regions 4045-4050 act as particleretention regions by trapping particles sets 4055-4057. Reagents can bepassed across the trapped particle sets by flowing the reagents throughsingle-depth wide channel regions 4060-4070. In one aspect, packets ofparticles are aligned to form trains of particles in double depthchannels 4045-4050.

FIG. 5 shows an embodiment adapted to sequencing. Microfluidic device500 comprises main channel 510 and reagent introduction channels 515-530(as depicted, these are coupled to reagents for separate sequencingreactions, e.g., comprising A, G, C, or T nucleotides. Sample train 531comprising a plurality of samples, e.g., particle sets 535-550, ispassed back and forth through intersections 560-590. Reagent fromchannels 515-530 is flowed across each sample (or selected samples) intrain 531 as the train passes the corresponding coupled intersection.Particle retention element 595 is an optional element.

FIG. 14 shows an additional device embodiment adapted to sequencing.Capillary 1405 (which is optionally a channel in a microfluidic device)comprises particle retention element 1410 and particle set 1415.Particle retention element 1410 comprises a particle set that is fixedor immobilized in capillary 1405, thus creating particle retentionregion 1420 in capillary 1405. In a sequencing reaction, a set ofparticles, e.g., coated with a DNA template, is optionally flowedthrough capillary 1405 from inlet region 1425. For example, inlet region1425 is optionally fluidly coupled to a sipper capillary, which duringoperation is fluidly coupled to a microwell plate. The set of DNA coatedparticles flows through capillary 1405 and is held or retained againstparticle retention element 1410, causing the set of DNA particles tobecome fixed in capillary 1405, such as particle set 1415. Sequencingreagents are then optionally flowed across particle set 1415 andoptionally rinsed from the channel between introductions of differentreagents. Particle retention element 1410 forms a porous barrier, whichstops particle set 1415, thus allowing, e.g., a series of reagents to beflowed across the DNA template and then rinsed away if desired, e.g.,four different nucleotides are serially flowed across the templatecoated particle set 1415 and unincorporated nucleotides are optionallyrinsed away between steps. At the same time, the pore size of particleretention element 1410 has a pore size allowing, e.g., liquid reagentsthrough so they are optionally rinsed out of capillary 1405, e.g., intoa waste reservoir proximal to downstream region 1430.

An alternate version of a particle retention element is shown in FIG.15. Particle retention element 1510 is sealed within housing 1540.Particle sets and/or liquid reagents are flowed through capillary 1505.Particle sets with a mean diameter greater than the pore size of theparticle retention element are stopped in capillary retention region1515 by particle retention element 1510 and liquid reagents and particlesets with a smaller mean diameter than the pore size of particleretention area are flowed past particle retention area 1515, e.g., by avacuum fluidly coupled to downstream capillary region 1530. In anotherembodiment, centrifugal force is used to draw reagents through capillary1505.

Alternatively, multiple solid phase components are flowed through andretained by a particle retention element. FIG. 16 shows particleretention element 1610 serving as a barrier to particle set 1615, whichserves as a barrier to particle set 1617, which serves as a barrier toparticle set 1619. Three particle sets are thus stacked in capillary1605. Particle sets 1615, 1617, and 1619 optionally have the same meandiameter or different mean diameters. Particle retention element 1610comprises a set of particles which when fixed within capillary 1605forms a porous barrier to the movement of other particle sets. Liquidreagents are optionally flowed through capillary 1605 to contactparticle sets 1615, 1617, and 1619. The liquid reagents are optionallyintroduced from a microwell plate via a sipper capillary fluidly coupledto upstream region 1625. The liquid reagents flow across and throughparticle sets 1615, 1617, and 1619 and react, e.g., with a DNA templateand primer on the particles, and flow through particle retention element1610, e.g., pulled by a vacuum fluidly coupled to downstream capillaryregion 1630.

FIG. 6 shows an embodiment adapted to high-throughput methods. Inparticular, a “sipper” (e.g., an electropipettor), e.g., sipper 610,draws one or more sets of beads or reagents ABCD and dispenses it intochannel 615 or into multiple channels, e.g., parallel assay channels,where the sets are sent as packet 620 across target beads or reagentsites, e.g., regions 625 or 630. This is faster than sipping beads orreagents individually and passing them across the relevant reactionsite.

FIG. 7 shows a multiplexing arrangement useful for DNA sequencing. Thebold straight lines represent fluid channels connected to a sipperchannel. The dotted lines represent electrical wires or pressureconduits connecting each of the reagent lines to a single controller. Inthis example, 3 sipper channels and 12 electrodes or pressure portsrequire only 4 controllers, instead of the more typical 12. An exampleof an assay using this arrangement is DNA sequencing, in which DNAsamples are on beads, one per sipper channel. The side channels havedifferent nucleotides that are passed over the beads sequentially. Allof the bead samples will have the same reagent stream pass across them.For example, multiple DNA samples, e.g., three different DNA templates,are simultaneously sequenced using only four controllers, one for eachreagent line and one for the main line. Alternatively, introduction ofreagents from a sipper capillary fluidly coupled to a microwell plateand to each of the main reaction channels in FIG. 7 allows the DNAsamples to be sequenced using only one controller coupled to each of thereaction channels.

FIG. 8 shows access to a particle library using one or more sippers. Inparticular, body 801 comprises particle channels 810-825 connected tosipper channel 830 which accesses a particle library (e.g., particlesets on a microwell plate). Packets of particles (e.g., comprisingnucleic acid templates for sequencing) are directed (e.g., byelectrokinesis or pressure-based mechanisms) into channels 810-825 toprovide an array of particle sets. For example, an electrical potentialor a vacuum is optionally applied at one or more of wells 835-850.Similarly, reagents (e.g., sequencing reagents) are flowed from any ofwells 860-875 through fine channel network 880 and into broad channel885, across the particle sets within channels 810-825, where thereagents interact with the particles in the channels. Alternatively, anindividual channel is used in place of fine channel network 880. Theparticles are first flowed within fine channel network 880 to providemore even flow of reagents into broad channel 885. Waste reagents areflowed into waste well 890. Flow is provided by applying electrical orpressure potentials at one or more of wells 860-875 and 890. Althoughillustrated for clarity as a single sipper channel arrangement, multiplesipper channels interconnected to microfluidic structures can also beused, e.g., for simultaneous and/or parallel access to samples orfluidic reagents. Alternatively, a train of reagents is flowed from amicrowell plate into sipper 830 and directly into each of channels810-825.

FIG. 9A shows an expanded cross-sectional view of particle packetswithin deep channels having an intersecting broad shallow channel. Inparticular, particle sets 900-910 are shown in channels 815-825, i.e.,the view is a cross section of a portion of FIG. 8 comprising thechannels. Flow of reagents is through broad channel 915, across particlesets 900-910. As depicted, body 801 comprises upper layer 930 and lowerlayer 940. FIG. 9B shows essentially the same elements in an expandedtop view, additionally depicting particle packets 950-975.

FIG. 10 shows sequential loading of particle sets or packets into achannel. Particle set 1005 is flowed through channel 1000 until blockedby particle retention element 1040. Over time, particle sets 1010-1020are accessed from a particle library, with each particle set abuttingthe previously loaded particle set(s). Particle sets 1005-1020 areoptionally the same or different sizes. Particle retention element 1040is optionally a sintered glass frit, glass, plastic, a fixed set ofparticles, e.g., epoxy coated particles, or the like. Typically,particle retention element 1040 is a porous barrier that has a pore sizesmaller than the mean diameter of the particle sets of interest, e.g.,particle sets 1005-1020.

FIG. 11, panel A, shows a parallel particle array in which sampleparticle sets 1105 are separated by blank particle sets 1110 in channels1115-1125. Panel B shows particle sets 1130-1145 in particle retentionregion 1150 abutting narrow channel region 1160. Panel C depicts analternate embodiment in which particle sets 1130-1145 are held inparticle retention region 1150 which abuts physical barrier 1155.

FIG. 12 shows a schematic integrated system of the invention. A computeror other microprocessing device directs materials from a sample systemto an array for analysis. If appropriate to the assay, array members aredirected from a particle library to the array to comprise or modify thearray. The selection of particle members is optionally in response to anassay signal from the array. In general, assay signals are directed fromthe array to a detector which detects the signals. The signalinformation is converted to a digital format and sent to the computer,which reads and/or stores the information. Optionally, the informationis used to select additional samples by or from the sampling systemand/or additional particle sets from the particle library.

For example, during operation of the integrated system illustrated inFIG. 12, an array is formed by accessing a particle library, followingan instruction set provided by the computer. The computer directs flowof fluidic (or particulate) reagents to the array through the samplingsystem (e.g., a system comprising a sipper channel which contacts andsips fluid from a relevant source of material, e.g., a micro titerplate). The fluidic reagents interact with the array members, providinga detectable signal. The signal is detected by the detector andconverted into digital information which is stored and/or manipulated bythe computer. Optionally, the digital information is used to provide thelogical basis for selecting additional array or fluidic or particulatereagents from the particle library and/or through the sampling system.In these cases, the computer selects the additional array or fluidic orparticulate reagent(s) and directs flow to the array and or modificationor movement of the array.

Particle Stacking

As noted above, the particle retention region can take any of a numberof forms in the present invention. For example, in one embodiment, afirst set of particles is flowed into a microfluidic region (e.g.,channel region, chamber, etc.) having a region with sufficiently smalldimension to inhibit movement of the first particle set. The firstparticle set stacks against the small dimensioned region of the channel.Subsequently, a second, third, fourth . . . nth set of particles can bemoved into the channel, where they will stack against, e.g., the firstparticle set. Even though the second . . . nth particle sets can besmall enough to pass through the small dimensioned region of thechannel, they are retained by stacking against the first particle set.Thus, the first particle set acts as a matrix preventing passage ofsubsequent particle sets. In this embodiment, each set of stackedparticles is larger in diameter than the typical voids between theparticles of the adjacent set. Thus, large retention particles can betrapped in a region of a microfluidic device and medium sized particlescan then be stacked next to the large particles, where the medium sizedparticles are larger in dimension than the voids between the largeparticles. Subsequently, small particles can be stacked next to themedium sized particles, where the small particles are larger in diameterthan the voids between the medium sized particles. Optionally, the smallparticles can be smaller than the voids between the large particles, asthey will be blocked from downstream flow by the medium sized particles.Even smaller particles can be stacked next to the small particles, etc.Of course, large particles can also be stacked next to fixed orotherwise retained small particles, as long as the large particlescannot pass between the voids of the stacked or fixed small particles.

For example, beads having a cross-section of about 100 μm to about 200μm are optionally used to form a fixed porous matrix or barrier in amicrochannel. The porous matrix is optionally used to capture or retainsmaller particles, e.g., particles comprising a cross-section of about30 μm to about 80 μm, e.g., a 40 μm particle set. Even smallerparticles, e.g., particles having a cross-section less than 30 μm, e.g.,about 5 μm to about 11 μm, are then captured and retained, e.g., by thesecond particle set, i.e., the 40 μm particle set.

There are several advantages to embodiments where particle sets areretained by other particle sets, rather than simply by e.g., physicaldimensions of the microfluidic system in which the particles are flowed.For example, the particle retention region is switchable in theseconfigurations, providing for dynamic construction and removal of thearrays and of the particle retention region (advantages of switchablearrays are set forth supra, including, e.g., creation of “smart” and“programmable” particle arrays). Another advantage to this configurationis that particles of extremely small dimensions can be used in thesecond . . . nth position, which increases the diversity of particletypes which are accessible by this approach.

Small particles also have properties which are, themselves, advantageousfor some embodiments. For example, several small particles havesignificantly more surface area than a single large particle thatoccupies the same volume as the smaller particles. This increase insurface area allows attachment/association of a greater number ofmolecules to the particle, increasing the density of the molecules ofinterest in the array. This is useful, e.g., for detecting assay signalswhich result from interaction with the molecules of interest, i.e., forincreasing the signal-to-noise ratio of assay signals in the assay.

For example, in one embodiment, a linear array of beads is made in amicrofluidic channel by stacking beads of a diameter larger than thedepth of the downstream channel created by a shelf, raised area,narrowed area, or other constriction within the channel (or othermicrofluidic structural element such as a chamber, cavity or the like).The size of the beads determines the surface area and binding capacityof the beads. The available binding capacity of the array is increasedby stacking beads of a smaller diameter adjacent to the larger diameterbeads, such that the diameter of the smaller diameter beads is largerthan the voids between the larger diameter beads. For example, in oneembodiment, a selected set of 4.4 micron beads have surface area of,roughly, 1.286*10¹² um²/g and the spaces between the beads areapproximately 1.8 microns. Packing beads of 2.5 microns next to the 4.4micron beads provides about 1.7 times as much surface area per unitvolume as the 4.4 micron beads. This procedure is optionally repeatedwith increasing surface area as the adjacent bead packages becomesmaller and smaller.

In a similar aspect, magnetic particles or affinity particles can beused to create particle retention regions for non-magnetic/non affinityparticles. In these embodiments, the first particle set (which is, e.g.,a magnetic particle (i.e., a particle which generates a magnetic fieldor which is attracted to a magnetic field), or an affinity particle) isflowed into the particle retention region where it is retained bymagnetic or affinity forces (e.g., covalent or non-covalent chemicalbonding between the particle or molecules disposed on the particle and aregion of the relevant microfluidic system). Second, third . . . nthparticle sets are flowed into contact with the fixed particle, where thefixed particle acts as a retention element to block flow of the fixedparticles. As above, small particles are typically stacked next to fixedor otherwise retained larger particles, e.g., where the small particlescannot pass between the voids of the stacked or fixed larger particles(of course, the small particles can be magnetic or affinity particles,with larger particles stacking against the fixed smaller particles aswell).

As described supra, particles can be essentially any size or shape. Inembodiments where arrays are made by stacking of adjacent sets ofparticles, it is desirable for sets to be of sufficient diameter thatthey cannot flow between adjacent particle sets. Thus, in one typicalaspect, the smallest dimension of a set of particles is larger than thevoids between the adjacent downstream particle set.

Number and Types of Array Members

The number of ordered sets constituting the array depends on theselected application. For example, as discussed in more detail herein,one exemplar array for sequencing nucleic acids comprises about 2, 3, or4 sets of particles (e.g., beads, cells, microspheres, etc.). In otherimplementations, 5, 10, 50, 100, 500, 1000, 5,000, 10,000, 50,000 oreven 100,00 or more different sets of particles can be present in thearrays. The precise number of particles in an array depends on theintended use of the array. For example, larger arrays are especiallyuseful, e.g., in screening molecular libraries against one or moretargets bound to the member particles of the particle sets. Smallerarrays can be used as to screen a smaller number of targets, as iscommon, e.g., in diagnostic applications, where one or a few targets(e.g., nucleic acids corresponding to various disease states, such asaltered levels or altered types of oncogene products, p53, presence ofinfectious organisms (HIV and other viruses, bacteria, etc.) aredesirably screened.

The array components (i.e., particles) of the arrays of the inventioncan be essentially any discreet material which can be flowed through amicroscale system. Example particles include beads and biological cells.For example, polymer beads (e.g., polystyrene, polypropylene, latex,nylon and many others), silica or silicon beads, clay or clay beads,ceramic beads, glass beads, magnetic beads, metallic beads, inorganiccompound beads, and organic compound beads can be used. An enormousvariety of particles are commercially available, e.g., those typicallyused for chromatography (see, e.g., the 1999 Sigma “Biochemicals andReagents for Life Sciences Research” Catalog from Sigma (Saint Louis,Mo.), e.g., pp. 1921-2007; The 1999 Suppleco “Chromatography Products”Catalogue, and others), as well as those commonly used for affinitypurification (e.g., Dynabeads™ from Dynal, as well as many derivatizedbeads, e.g., various derivatized Dynabeads™ (e.g., the various magneticDynabeads™, which commonly include coupled reagents) supplied e.g., byPromega, the Baxter Immunotherapy Group, and many other sources).

A wide variety of particles useful in the present invention includethose used as components of sieving and molecular partition matrixes inthe art. Many such matrixes are available, and can be used to constituteparticle arrays in the apparatus of the invention. For example, avariety of sieving matrixes, partition matrixes and the like areavailable from Supelco, Inc. (Bellefonte, Pa.; see, e.g., the 1997 (orlater) Suppleco catalogue). Common matrixes which are useful in thepresent invention include those generally used in low pressure liquidchromatography, gel electrophoresis and other liquid phase separations.Matrix materials designed primarily for non-liquid phase chromatographyare also useful in certain contexts, as the materials often retainparticulate characteristics when suspended in fluids. For a discussionof electrophoresis matrixes see, e.g., Weiss (1995) Ion ChromatographyVCH Publishers Inc.; Baker (1995) Capillary Electrophoresis John Wileyand Sons; Kuhn (1993) Capillary Electrophoresis: Principles and PracticeSpringer Verlag; Righetti (1996) Capillary Electrophoresis in AnalyticalBiotechnology CRC Press; Hill (1992) Detectors for CapillaryChromatography John Wiley and Sons; Gel Filtration: Principles andMethods (5th Edition) Pharmacia; Gooding and Regnier (1990) HPLC ofBiological Macromolecules Methods and Applications (Chrom. Sci. Series,volume 51) Marcel Dekker and Scott (1995) Techniques and Practices ofChromatography Marcel Dekker, Inc.

Commercially available low pressure liquid chromatography media suitableas particulate material (i.e., material for making particle sets) in avariety of applications include, e.g., non-ionic macroreticular andmacroporous resins which adsorb and release components based uponhydrophilic or hydrophobic interactions, such as Amberchrom resins(highly cross-linked styrene/divinylbenzene copolymers suitable forseparation of peptides, proteins, nucleic acids, antibiotics,phytopharmacologicals, and vitamins); the related Amberlite XAD seriesresins (polyaromatics and acrylic esters) and amberchroms (polyaromaticand polymethacrylates) (manufactured by Rohm and Haas, available throughSuppleco); Diaion (polyaromatic or polymethacrylic beads); Dowex(polyaromatics or substituted hydrophilic functionalized polyaromatics)(manufactured by Dow Chemical, available through Suppleco); Duolite(phenol-formaldehyde with methanolic functionality), MCI GEL sephabeads,supelite DAX-8 (acrylic ester) and Supplepak (polyaromatic) (all of thepreceding materials are available from Suppleco). For a description ofuses for Amberlite and Duolite resins, see, Amberlite/Duolite AnionExchange Resins (Available from Suppleco, 1997 Cat No. T412141). Gelfiltration chromatography matrixes are also suitable, includingsephacryl, sephadex, sepharose, superdex, superose, toyopearl, agarose,cellulose, dextrans, mixed bead resins, polystyrene, nuclear resins,DEAE cellulose, Benzyl DEA cellulose, TEAE cellulose, and the like(Suppleco).

Gel electrophoresis media comprising particulate material useful inmaking particle sets include silica gels such as Davisil Silica, E.Merck Silica Gel, Sigma-Aldrich Silica Gel (all available from Suppleco)in addition to a wide range of silica gels available for variouspurposes as described in the Aldrich catalogue/handbook (AldrichChemical Company (Milwaukee, Wis.)). Preferred gel materials includeagarose based gels, various forms of acrylamide based gels (reagentsavailable from, e.g., Suppleco, SIGMA, Aldrich, SIGMA-Aldrich and manyother sources) colloidal solutions such as protein colloids (gelatins)and hydrated starches.

A variety of affinity media for purification and separation of molecularcomponents are also available, including a variety of modified silicagels available from SIGMA, Aldrich and SIGMA-Aldrich, as well asSuppleco, such as acrylic beads, agarose beads, Mono beads, cellulose,sepharose, sepharose CL, toyopearl or the like chemically linked to anaffinity ligand such as a biological molecule. A wide variety ofactivated matrixes, amino acid resins, avidin and biotin resins,carbohydrate resins, dye resins, glutathione resins, hydrophobic resins,immunochemical resins, lectin resins, nucleotide/coenzyme resins,nucleic acid resins, and specialty resins are available, e.g., fromSuppleco, SIGMA, Aldrich or the like. See also, Hermanson et al. (1992)Immobilized Affinity Ligand Techniques Academic Press.

Other particulate media commonly used, e.g., in chromatography are alsoadaptable to the present invention, including activated aluminas,carbopacks, carbosieves, carbowaxes, chromosils, DEGS, Dexsil, Durapak,Molecular Sieve, OV phases, porous silica, chromosorb series packs,HayeSep series, Porapak series, SE-30, Silica Gel, SP-1000, SP-1200,SP-2100, SP-2250, SP-2300, SP2401, Tenax, TCEP, supelcosil LC-18-S andLC-18-T, Methacrylate/DVBm, polyvinylalcohols, napthylureas, non-polarmethyl silicone, methylpolysiloxane, poly(ethylene glycol)biscyanopropylpolysiloxane and the like.

Ion exchange chromatography resins comprising particulate material arecommercially available, including from EM Separations (Gibbstown, N.J.),BioSepra (Marlborough, Mass.), Polymer Laboratories (Amherst, Mass.),Perseptive Biosystems (Cambridge, Mass.), Toso Haas (Montgomeryville,Pa.) and Pharmacia (Uppsala, Sweden).

As noted herein, the definition for particles as intended hereinincludes both biological and non-biological particle material. Thus,cells are included within the definition of particles for purposes ofthe present invention.

Cell based microfluidic assays are described in a variety ofpublications by the inventors and their co-workers, including, Parce etal. “High Throughput Screening Assay Systems in Microscale FluidicDevices” WO 98/00231 and Knapp et al. “Closed Loop BiochemicalAnalyzers” (WO 98/45481; PCT/US98/06723). It is expected that one ofskill is fully able to culture cells and introduce them intomicrofluidic systems. In addition to Parce et al. and Knapp et al., manyreferences are available for the culture and production of many cells,including cells of bacterial, plant, animal (especially mammalian) andarchebacterial origin. See e.g., Sambrook, Ausubel, and Berger (allsupra), as well as Freshney (1994) Culture of Animal Cells, a Manual ofBasic Technique, third edition Wiley-Liss, New York and the referencescited therein, Humason (1979) Animal Tissue Techniques, fourth editionW.H. Freeman and Company; Ricciardelli, et al., (1989) In Vitro CellDev. Biol. 25:1016-1024; Payne et al. (1992) Plant Cell and TissueCulture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.(Payne); Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and OrganCulture; Fundamental Methods Springer Lab Manual, Springer-Verlag(Berlin Heidelberg N.Y.) (Gamborg); and Atlas and Parks (eds) TheHandbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.(Atlas). Additional information for plant cell culture is found inavailable commercial literature such as the Life Science Research CellCulture Catalogue (1998) from Sigma-Aldrich, Inc (St Louis, Mo.)(Sigma-LSRCCC) and, e.g., the Plant Culture Catalogue and supplement(1997) also from Sigma-Aldrich, Inc (St Louis, Mo.) (Sigma-PCCS). Oneparticularly preferred use for cell-based microfluidic assays is toscreen binding and/or internalization of cell ligands, e.g., cellreceptor ligands, drugs, co-factors, etc. This screening is considerablyfacilitated by arraying different cell sets into arrays of cells, whichcan then have reagent trains comprising any factor to be tested for invitro cellular activity flowed across the cell sets. Of course, cellscan also be present in reagent trains and passed into contact with otherarray members.

Cells can exist as sets of particles in a variety of formats in thepresent invention. For example, cells can be fixed to solid supportssuch as beads or other microparticles. Thus, arrays of the invention caninclude heterogeneous particles comprising solid supports and cells orother components of interest. Cells can also be trapped using strategiessimilar to those described herein for particles generally, i.e., byphysical trapping mechanisms. In addition, as cells comprise surfaceproteins and other molecules, it is convenient to fix cell bindingmolecules (cell receptor ligands, cell wall binding molecules,antibodies, etc.) either to regions of the channels of the microfluidicdevice, or to particles which are then fixed or localized in position bythe methods described herein.

The array particles can have essentially any shape, e.g., spherical,helical, spheroid, rod-shaped, cone-shaped, cubic, polyhedral, or acombination thereof (of course they can also be irregular, as is thecase for cell-based particles). In addition, the particles can be avariety of sizes. Typically, the particles are about 0.1 μm to about 500μm. Alternatively, the particles are about 0.5 μm to about 50 μm orabout 1 μm to about 20 μm. Particles are optionally coupled to reagents,affinity matrix materials, or the like, e.g., nucleic acid synthesisreagents, peptide synthesis reagents, polymer synthesis reagents,nucleic acids, nucleotides, nucleobases, nucleosides, peptides, aminoacids, monomers, cells, biological samples, synthetic molecules, orcombinations thereof. Particles optionally serve many purposes withinthe arrays, including acting as blank particles, dummy particles,calibration or marker particles, capture devices for low concentrationreagents, sample particles, reagent particles and test particles.

Linking Chemistries

The particles within the arrays of the invention can present a solid orsemi-solid surface for any of a variety of linking chemistries, allowingthe incorporation of biological and chemical components of interest intothe particle members of the arrays. A wide variety of organic andinorganic polymers, both natural and synthetic may be employed as thematerial for the solid surface. Illustrative polymers includepolyethylene, polypropylene, poly(4-methylbutene), polystyrene,polymethacrylate, poly(ethylene terephthalate), rayon, nylon, poly(vinylbutyrate), polyvinylidene difluoride (PVDF), silicones,polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and thelike. Other materials that may be employed include papers, ceramics,such as glass, metals, metalloids, semiconductive materials, cements, orthe like. In addition, substances that form gels, such as proteins(e.g., gelatins), lipopolysaccharides, silicates, agarose and are alsooptionally used.

A wide variety of linking chemistries are available for linkingmolecules to a wide variety of solid or semi-solid particle supportelements. These chemistries can be performed in situ (i.e., in themicrofluidic system, by flowing appropriate reagents, e.g., nucleicacids, proteins, and samples present in low concentrations, into contactwith the particles, or vice-versa), or outside of a microfluidicenvironment, e.g., prior to introduction of the particles into themicrofluidic system. It is impractical and unnecessary to describe allof the possible known linking chemistries for linking molecules to asolid support. It is expected that one of skill can easily selectappropriate chemistries, depending on the intended application.

In one preferred embodiment, the particles of the invention comprisesilicate elements (e.g., glass or silicate beads). An array ofsilicon-based molecules appropriate for functionalizing surfaces arecommercially available. See, for example, Silicon Compounds Registry andReview, United Chemical Technologies, Bristol, Pa. Additionally, the artin this area is very well developed and those of skill will be able tochoose an appropriate molecule for a given purpose. Appropriatemolecules can be purchased commercially, synthesized de novo, or it canbe formed by modifying an available molecule to produce one having thedesired structure and/or characteristics.

The substrate linker attaches to the solid substrate through any of avariety of chemical bonds. For example, the linker is optionallyattached to the solid substrate using carbon-carbon bonds, for examplevia substrates having (poly)trifluorochloroethylene surfaces, orsiloxane bonds (using, for example, glass or silicon oxide as the solidsubstrate). Siloxane bonds with the surface of the substrate are formedin one embodiment via reactions of derivatization reagents bearingtrichlorosilyl or trialkoxysilyl groups. The particular linking group isselected based upon, e.g., its hydrophilic/hydrophobic properties wherepresentation of an attached polymer in solution is desirable. Groupswhich are suitable for attachment to a linking group include amine,hydroxyl, thiol, carboxylic acid, ester, amide, isocyanate andisothiocyanate. Preferred derivatizing groups includeaminoalkyltrialkoxysilanes, hydroxyalkyltrialkoxysilanes,polyethyleneglycols, polyethyleneimine, polyacrylamide, polyvinylalcoholand combinations thereof.

By way of example, the reactive groups on a number of siloxanefunctionalizing reagents can be converted to other useful functionalgroups:

-   -   1. Hydroxyalkyl siloxanes (Silylate surface, functionalize with        diborane, and H2O2 to oxidize the alcohol);        -   a. allyl trichlorosilane→→3-hydroxypropyl        -   b. 7-oct-1-enyl trichlorchlorosilane→→8-hydroxyoctyl    -   2. Diol (dihydroxyalkyl) siloxanes (silylate surface and        hydrolyze to diol)        -   a. (glycidyl            trimethoxysilane→→(2,3-dihydroxypropyloxy)propyl    -   3. Aminoalkyl siloxanes (amines requiring no intermediate        functionalizing step)        -   a. 3-aminopropyl trimethoxysilane→aminopropyl    -   4. Dimeric secondary aminoalkyl siloxanes        -   a.            bis(3-trimethoxysilylpropyl)amine→bis(silyloxylpropyl)amine.            See, for example, Leyden et al., Symposium on Silylated            Surfaces, Gordon & Breach 1980; Arkles, Chemtech 7, 766            (1977); and Plueddemann, Silane Coupling Reagents, Plenum,            N.Y., 1982. These examples are illustrative and do not limit            the types of reactive group interconversions which are            useful in conjunction with the present invention. Additional            starting materials and reaction schemes will be apparent to            those of skill in the art.

The components which can be attached to a derivatized particle surfaceinclude peptides, nucleic acids, mimetics, large and small organicmolecules, polymers and the like. For example, moieties bearing apermanent charge or a pH dependent charge are useful in practicing thepresent invention. For example, the charged group can be a carboxylate,quaternary amine or protonated amine that is a component of an aminoacid that has a charged or potentially charged side chain. The aminoacids can be either those having a structure which occurs naturally orthey can be of unnatural structure (i.e., synthetic). Useful naturallyoccurring amino acids include, arginine, lysine, aspartic acid andglutamic acid. Surfaces utilizing a combination of these amino acids arealso of use in the present invention. Further, peptides comprising oneor more residues having a charged or potentially charged side chain areuseful coating components and they can be synthesized utilizingarginine, lysine, aspartic acid, glutamic acid and combinations thereof.Useful unnatural amino acids are commercially available or can besynthesized utilizing art-recognized methodologies. In those embodimentsin which an amino acid moiety having an acidic or basic side chain isused, these moieties can be attached to a surface bearing a reactivegroup through standard peptide synthesis methodologies or easilyaccessible variations thereof. See, for example, Jones, Amino Acid andPeptide Synthesis, Oxford University Press, Oxford, 1992. In addition,nucleic acids attached to a particle surface are optionally sequenced orused as a calibration particle or marker.

Linking groups can also be placed on the particles of the invention.Linking groups of use in the present invention can have a range ofstructures, substituents and substitution patterns. They can, forexample be derivatized with nitrogen, oxygen and/or sulfur containinggroups which are pendent from, or integral to, the linker groupbackbone. Examples include, polyethers, polyacids (polyacrylic acid,polylactic acid), polyols (e.g., glycerol), polyamines (e.g., spermine,spermidine) and molecules having more than one nitrogen, oxygen and/orsulfur moiety (e.g., 1,3-diamino-2-propanol, taurine). See, for example,Sandler et al. Organic Functional Group Preparations 2nd Ed., AcademicPress, Inc. San Diego 1983. A wide range of mono-, di- andbis-functionalized poly(ethyleneglycol) molecules are commerciallyavailable and will prove generally useful in this aspect of theinvention. See, for example, 1997-1998 Catalog, Shearwater Polymers,Inc., Huntsville, Ala. Additionally, those of skill in the art haveavailable a great number of easily practiced, useful modificationstrategies within their synthetic arsenal. See, for example, Harris,Rev. Macromol. Chem. Phys., C25(3), 325-373 (1985); Zalipsky et al.,Eur. Polym. J., 19(12), 1177-1183 (1983); U.S. Pat. No. 5,122,614,issued Jun. 16, 1992 to Zalipsky; U.S. Pat. No. 5,650,234, issued toDolence et al. Jul. 22, 1997, and references therein.

In a preferred embodiment of the invention, the coupling chemistries forcoupling materials to the particles of the invention arelight-controllable, i.e., utilize photo-reactive chemistries. The use ofphoto-reactive chemistries and masking strategies to activate couplingof molecules to substrates, as well as other photo-reactive chemistriesis generally known (e.g., for semi-conductor chip fabrication and forcoupling bio-polymers to solid phase materials). The use ofphoto-cleavable protecting groups and photo-masking permits typeswitching of both mobile and fixed array members, i.e., by altering thepresence of substrates present on the array members (i.e., in responseto light). Among a wide variety of protecting groups which are usefulare nitroveratryl (NVOC)-methylnitroveratryl (Menvoc), allyloxycarbonyl(ALLOC), fluorenylmethoxycarbonyl (FMOC),-methylnitro-piperonyloxycarbonyl (MeNPOC), —NH-FMOC groups, t-butylesters, t-butyl ethers, and the like. Various exemplary protectinggroups (including both photo-cleavable and non-photo-cleavable groups)are described in, for example, Atherton et al., (1989) Solid PhasePeptide Synthesis, IRL Press, and Greene, et al. (1991) ProtectiveGroups In Organic Chemistry, 2nd Ed., John Wiley & Sons, New York, N.Y.,as well as, e.g., Fodor et al. (1991) Science, 251: 767-777, Wang (1976)J. Org. Chem. 41: 3258; and Rich, et al. (1975) J. Am. Chem. Soc. 97:1575-1579. The use of these and other photo-cleavable linking groups fornucleic acid and peptide synthesis on solid supports is awell-established methodology.

In one useful variation of these methods, solid phase arrays are adaptedfor the rapid and specific detection of multiple polymorphicnucleotides. Typically, a nucleic acid probe is chemically linked to asolid support and a target nucleic acid (e.g., an RNA or correspondingamplified DNA) is hybridized to the probe. Either the probe, or thetarget, or both, can be labeled, typically with a fluorophore. Where thetarget is labeled, hybridization is detected by detecting boundfluorescence. Where the probe is labeled, hybridization is typicallydetected by quenching of the label by the bound nucleic acid. Where boththe probe and the target are labeled, detection of hybridization istypically performed by monitoring a signal shift such as a change incolor, fluorescent quenching, or the like, resulting from proximity ofthe two bound labels. In some assay formats, the above format isinverted, with expression products being fixed to array members andprobes being moved into contact with the array.

In another variation, solid-phase arrays are used to detect samples evenat very low concentrations. Particles and particle arrays are veryefficient at capturing molecules in a fluid stream. Therefore, theyprovide suitable molecule capture devices for studying systems in whicha sample or reagent is in very low concentration, e.g., in single cellRNA analysis. Typical methods for studying such systems involve DNAarrays having the non-precious reagents, e.g., those available in largeor adequate quantities and concentrations, spread out in definedlocations across a substrate. The precious or low concentration sample,e.g., cell contents or amplification product, is typically diffusedacross the array, e.g., to find a hybridization partner. By attachingthe precious or low concentration sample to a particle or particlearray, it can be placed in a defined location and then non-precioussamples are optionally flowed across the array, allowing detection ofthe sample at very low concentrations. For example, a few cells areoptionally flowed through a capillary comprising a particle array, e.g.,beads, e.g., hybridized with a capture reagent. The cells are lysed andthe mRNA from the cells is captured on the particles. The capillary isthen optionally used to flow reagents across the particles, e.g.,hybridization reagents that bind, e.g., specifically, to the mRNA ofinterest, if it is present. The capillary is rinsed after each exposureand any resulting hybridization is detected, e.g., by fluorescencedetection. Alternatively, the captured molecule is amplified and theproduct captured, e.g., immediately or in about the same vicinity toenhance the signal before bringing hybridization or probe reagentsacross the capture area. In other aspects, different probe reagentscomprising different fluorophores are mixed to detect several componentsat once. Alternatively, capillary or channel surfaces are used tocapture the low concentration molecule instead of particles.

In other aspects, the particles of the present invention are used asmarker particles or calibration particles. For example, charged beads,e.g., to which a dye is attached, are optionally used as markers incapillary electrophoresis, especially when very low mobility isrequired. For example, a marker that flows slower than any sample isoptionally used. Alternatively, a neutral particle or bead is used withan attachment, functional group, or linker that is charged. Typicalmolecules for use on the particles include, but are not limited to,molecules that are similar to the sample of interest. For example, anucleic acid is preferably used in electrophoresis of nucleic acids. Atypical DNA or RNA molecule attached to such a marker or calibrationbead comprises about 10 thousand base-pairs to about 20 thousand basepairs, e.g., a 17 thousand base pair nucleic acid. The marker particleor bead is used, for example, to determine the position of one or moremember of an array. For example, the marker is optionally used todetermine the position of an array member that has captured a lowconcentration sample as described above. The particle or bead typicallycomprises a charged moiety or particle and a label moiety, e.g., afluorescent dye or a charged label moiety. The label moiety isoptionally detected, e.g., by fluorescence, to determine and calibratepositions of array members.

In one embodiment of this concept, an array of probes are synthesized onsolid support particles constituting array members. Using typical arraymasking technologies and photoprotective chemistry, it is possible togenerate ordered arrays of nucleic acid probes with large numbers ofprobes. In the embodiments of the present invention, even photomaskingis unnecessary, making photoprotective chemistry particularly useful. Inparticular, the array members can be flowed past a light source in aselected order in the presence of selected reagents, permittingselective addition of components to the array, without actuallyperforming chip masking. Of course, however, chip masking strategies canalso be used, i.e., array members can be fixed in place and selectivelyexposed to light. Either method is used, for example, to place aprecious sample, e.g., a sample that is only available in small amounts,in a defined location and detect it at very low concentrations.

In brief, a combinatorial strategy allows for the synthesis of arrayscontaining a large number of different elements such as fixed nucleicacids, using a minimal number of synthetic steps. For instance, ingeneral in solid-phase masking technologies, it is possible tosynthesize and attach all possible DNA 8-mer oligonucleotides (48, or65,536 possible combinations) using only 32 chemical synthetic steps. Ingeneral, these procedures provide a method of producing 4n differentoligonucleotide probes on an array using only 4n synthetic steps.Alternatively, due to the high-throughput reaction speeds ofmicrofluidic systems, it is possible to perform large numbers ofreactions with linear or parallel fluidic manipulations in feasiblyshort periods of time.

As noted, light-directed combinatorial synthesis of oligonucleotidearrays on glass array members is performed with automatedphosphoramidite chemistry and, optionally, chip masking techniquessimilar to photoresist technologies in the computer chip industry.Typically, a glass surface is derivatized with a silane reagentcontaining a functional group, e.g., a hydroxyl (for nucleic acidarrays) or amine group (for peptide or peptide nucleic acid arrays)blocked by a photolabile protecting group. Photolysis through aphotolithographic mask, or by selective flow, e.g., in a microfluidicsystem past a light source, is used selectively to expose functionalgroups which are then ready to react with incoming photoprotectedelements (e.g., for nucleic acids, 5′-photoprotected nucleosidephosphoramidites). The photoprotected elements react with those siteswhich are illuminated (and thus exposed by removal of the photolabileblocking group). Thus, e.g., phosphoramidites only add to those areasselectively exposed from the preceding step. These steps are repeateduntil the desired array of sequences has been synthesized on the arrayparticle. Combinatorial synthesis of different molecules at differentlocations on the array is determined by the pattern of illuminationduring synthesis, relative to the array (again, the array can be mobileor fixed in the present invention) and the order of addition of couplingreagents. Monitoring of hybridization of target elements to the array istypically performed with fluorescence microscopes, laser scanningmicroscopes, CCD arrays or the like.

Although light-directed coupling chemistries are preferred, anddescribed above in some detail for exemplary purposes, these are not theonly feasible routes to producing arrays with a variety of differentparticle members. The use of microfluidic fluid movement to movereagents into contact with array members can also be used. Inparticular, solid surfaces are derivatized as noted above in preparationfor addition of components such as nucleotide synthesis reagents,peptides, or the like. Between coupling steps, protective groups can beused as noted above. Instead of photoprotective cleavage, other cleavageagents can be used, depending on the nature of the reaction, e.g.,acids, bases, or the like. Indeed, because it is possible to flow onlydesired reagents into contact with selected array members, it is notnecessary to use blocking groups at all. The elimination of blockinggroups is one of the many advantages of the present invention overstandard solid phase synthesis techniques.

Array Templates

The array or reagents contacting the array can involve templatehybridization reagents or the like, including a first nucleic acid whichis fully or partially complementary to a second nucleic acid complexedwith a particle set of the array, a first protein which specificallyhybridizes to one or more component with a particle set of the array, afirst antibody which specifically hybridizes to one or more componentwith a particle set of the array, a hybridization buffer, a blockingreagent, and a labeled probe nucleic acid. The methods optionallycomprise flowing liquid reagents into contact with one or more arraymember and detecting the resulting hybridization of the liquid reagentto the array member.

Sequencing and PCR in Microfluidic Systems

In a preferred embodiment of the invention, the microarrays of theinvention are used for sequencing nucleic acids. The devices of theinvention optionally include reagents (which may be part of the array orflowed into contact with the array, e.g. in a reagent train) forperforming a biological or chemical assay. The liquid reagent or arraycan include a nucleic acid sequencing reagent such as a liquid solutioncomprising a nucleotide, a liquid solution comprising a polymerase, aliquid solution comprising a dNTP, a ddNTP, a dNTP analog, or afluorescent dNTP, a liquid solution comprising a sufurylase, a liquidsolution comprising an apyrase, a liquid solution comprising inorganicphosphate, a liquid solution comprising ATP, a liquid solutioncomprising a thermostable polymerase, a liquid solution comprising anendonuclease, a liquid solution comprising an exonuclease, a liquidsolution comprising a phosphatase, a liquid solution comprising anintercalator, a liquid solution comprising a reducing agent, a liquidsolution comprising Mg⁺⁺, a liquid solution comprising a molecularcrowding agent, e.g., PEG, a liquid solution comprising a buffer, aliquid solution comprising a salt, a salt, DTT, BSA, a detergent (e.g.,triton or tween), chemicals to inhibit or enhance electroosmotic flow(e.g., polyacrylamide) or the like.

Standard Chain Termination Sequencing

Most DNA sequencing today is still carried out by chain terminationmethods of DNA sequencing. The most popular chain termination methods ofDNA sequencing are variants of the dideoxynucleotide mediated chaintermination method of Sanger. See, Sanger et al. (1977) Proc. Nat. Acad.Sci., USA 74:5463-5467. For a simple introduction to dideoxy sequencing,see, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (Supplement 38, current through 1998)(Ausubel), Chapter 7. Thousands of laboratories employ dideoxynucleotidechain termination techniques. Commercial kits containing the reagentsmost typically used for these methods of DNA sequencing are availableand widely used. These methods of DNA sequencing are adaptable to thearrays of the invention. In particular, array members can comprise e.g.,template nucleic acids, e.g., chemically coupled or hybridized toparticle surfaces. Reagent trains comprising sequencing reagents arepassed across the template nucleic acids (e.g., using electrophoresis,or electroosmotic or pressure-based reagent flow) where they contact thetemplates. Reaction products can be analyzed directly, or followingdissociation and electrophoresis within the microfluidic system.

In addition to the Sanger methods of chain termination, new PCRexonuclease digestion methods have also been developed for DNAsequencing. Direct sequencing of PCR generated amplicons by selectivelyincorporating boronated nuclease resistant nucleotides into theamplicons during PCR and digestion of the amplicons with a nuclease toproduce sized template fragments has been performed (Porter et al.(1997) Nucleic Acids Research 25(8):1611-1617). In the methods, 4 PCRreactions on a template are performed, in each of which one of thenucleotide triphosphates in the PCR reaction mixture is partiallysubstituted with a 2′ deoxynucleoside 5′-∀[P-borano]-triphosphate. Theboronated nucleotide is stochastically incorporated into PCR products atvarying positions along the PCR amplicon in a nested set of PCRfragments of the template. An exonuclease which is blocked byincorporated boronated nucleotides is used to cleave the PCR amplicons.The cleaved amplicons are then separated by size using polyacrylamidegel electrophoresis, providing the sequence of the amplicon. Anadvantage of this method is that it requires fewer biochemicalmanipulations than performing standard Sanger-style sequencing of PCRamplicons. These methods are similarly adaptable to the arrays andmicrofluidic systems of the invention. In particular, PCR can beperformed by heating and cooling all or part of a microfluidic system.

It is expected that one of skill is familiar with fundamental sequencingmethodologies applicable to the present invention. Examples oftechniques for making and sequencing nucleic acids, and instructionssufficient to direct persons of skill through most standard cloning andother template preparation exercises are found in Berger and Kimmel,Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989)Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook); and CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (1997, supplement 37) (Ausubel). Basicprocedures for cloning and other aspects of molecular biology andunderlying theoretical considerations are also found in Lewin (1995)Genes V Oxford University Press Inc., NY (Lewin); and Watson et al.(1992) Recombinant DNA Second Edition Scientific American Books, NewYork. Product information from manufacturers of biological reagents andexperimental equipment also provide information useful in knownbiological methods. Such manufacturers include the Sigma ChemicalCompany (Saint Louis, Mo.); New England Biolabs (Beverly, Mass.); R&Dsystems (Minneapolis, Minn.); Pharmacia LKB Biotechnology (Piscataway,N.J.); CLONTECH Laboratories, Inc. (Palo Alto, Calif.); ChemGenes Corp.,(Waltham Mass.) Aldrich Chemical Company (Milwaukee, Wis.); GlenResearch, Inc. (Sterling, Va.); GIBCO BRL Life Technologies, Inc.(Gaithersberg, Md.); Fluka Chemica-Biochemika Analytika (Fluka ChemieAG, Buchs, Switzerland); Invitrogen (San Diego, Calif.); Perkin Elmer(Foster City, Calif.); and Strategene; as well as many other commercialsources known to one of skill.

In one aspect, the generation of large nucleic acids is useful inpracticing the invention, e.g., as templates fixed to array members,e.g., for sequencing long regions of nucleic acids, or for monitoringexpression products by hybridization of biological materials to thefixed templates. It will be appreciated that such templates areparticularly useful in some aspects where the methods and devices of theinvention are used to sequence large regions of DNA, e.g., for genomicstypes of applications. An introduction to large clones such as YACs,BACs, PACs and MACs as artificial chromosomes is provided by Monaco andLarin (1994) Trends Biotechnol 12 (7): 280-286.

The construction of nucleic acid libraries of template nucleic acids isdescribed in the above references. YACs and YAC libraries are furtherdescribed in Burke et al. (1987) Science 236:806-812. Gridded librariesof YACs are described in Anand et al. (1989) Nucleic Acids Res. 17,3425-3433, and Anand et al. (1990) Nucleic Acids Res. Riley (1990)18:1951-1956 Nucleic Acids Res. 18(10): 2887-2890 and the referencestherein describe cloning of YACs and the use of vectorettes inconjunction with YACs. See also, Ausubel, chapter 13. Cosmid cloning isalso well known. See, e.g., Ausubel, chapter 1.10.11 (supplement 13) andthe references therein. See also, Ish-Horowitz and Burke (1981) NucleicAcids Res. 9:2989-2998; Murray (1983) Phage Lambda and Molecular Cloningin Lambda II (Hendrix et al., eds) 395-432 Cold Spring HarborLaboratory, NY; Frischauf et al. (1983) J. Mol. Biol. 170:827-842; and,Dunn and Blattner (1987) Nucleic Acids Res. 15:2677-2698, and thereferences cited therein. Construction of BAC and P1 libraries is wellknown; see, e.g., Ashworth et al. (1995) Anal Biochem 224 (2): 564-571;Wang et al. (1994) Genomics 24(3): 527-534; Kim et al. (1994) Genomics22(2): 336-9; Rouquier et al. (1994) Anal Biochem 217(2): 205-9; Shizuyaet al. (1992) Proc Natl Acad Sci USA 89(18): 8794-7; Kim et al. (1994)Genomics 22 (2): 336-9; Woo et al. (1994) Nucleic Acids Res 22(23):4922-31; Wang et al. (1995) Plant (3): 525-33; Cai (1995) Genomics 29(2): 413-25; Schmitt et al. (1996) Genomics 1996 33(1): 9-20; Kim et al.(1996) Genomics 34(2): 213-8; Kim et al. (1996) Proc Natl Acad Sci USA(13): 6297-301; Pusch et al. (1996) Gene 183(1-2): 29-33; and, Wang etal. (1996) Genome Res 6(7): 612-9.

In general, where the desired goal of a sequencing project is thesequencing of a genome or expression profile of an organism, a libraryof the organism's cDNA or genomic DNA is made according to standardprocedures described, e.g., in the references above. Individual clonesare isolated and sequenced, and overlapping sequence information isordered to provide the sequence of the organism. See also, Tomb et al.(1997) Nature 539-547 describing the whole genome random sequencing andassembly of the complete genomic sequence of Helicobacter pylori;Fleischmann et al. (1995) Science 269:496-512 describing whole genomerandom sequencing and assembly of the complete Haemophilus influenzaegenome; Fraser et al. (1995) Science 270:397-403 describing whole genomerandom sequencing and assembly of the complete Mycoplasma genitaliumgenome and Bult et al. (1996) Science 273:1058-1073 describing wholegenome random sequencing and assembly of the complete Methanococcusjannaschii genome.

Recently, Hagiwara and Curtis (1996) Nucleic Acids Research24(12):2460-2461 developed a “long distance sequencer” PCR protocol forgenerating overlapping nucleic acids from very large clones tofacilitate sequencing, and methods of amplifying and tagging theoverlapping nucleic acids into suitable sequencing templates. Themethods can be used in conjunction with shotgun sequencing techniques toimprove the efficiency of shotgun methods typically used in wholeorganism sequencing projects. As applied to the present invention, thetechniques are useful for identifying and sequencing genomic nucleicacids using the arrays of the present invention. In particular, one ormore component of the PCR reactions, YACs, vectorettes, or the like usedin the long distance sequencer method are fixed to an array, and thearray performed in a microfluidic system by flowing other components ofthe long distance sequencer method into contact with the fixed componenton the array. This method particularly benefits from the use of arraysdue to the need to organize several reactions simultaneously for thelong distance sequencer method. Products can be assayed in parallel orsequentially, facilitating the selection of subsequent reactioncomponents.

Sequencing by Incorporation/Synthesis

In a preferred embodiment, the present invention provides for sequencingby synthesis or incorporation. A number of basic sequencing byincorporation methods are known, e.g., as set forth in Hyman U.S. Pat.No. 4,971,903; Malemede U.S. Pat. No. 4,863,849; Cheeseman U.S. Pat. No.5,302,509, and Canard U.S. Pat. No. 5,798,210. Generally, any detectableevent associated with incorporation of a nucleotide can be used tomonitor sequencing reactions. In sequencing by incorporation methods,incorporation of nucleotide reagents into nucleic acids (typically by ausing a polymerase to extend a primer hybridized to a complementarytemplate nucleic acid) is monitored to provide an indication of thesequence of a template nucleic acid. This can be performed byselectively adding reagents comprising labels such as bases comprisingfluorescent moieties, e.g., four detectably different fluorescentmoieties, to e.g., a member of an array set and monitoring incorporationof the label into the nucleic acid.

A variety of nucleotides which have fluorescent labels can be added in abase specific fashion by a polymerase. For example, Hawkins et al. U.S.Pat. No. 5,525,711 describe pteridine nucleotide analogs for use influorescent DNA probes. These analogs can be incorporated by, e.g., Taqpolymerase, sequenase, DNA polymerase Klenow fragment, or the like.

In both sequencing methodologies and elsewhere herein, it will berecognized that fluorescent labels are not to be limited to singlespecies organic molecules, but include inorganic molecules,multi-molecular mixtures of organic and/or inorganic molecules,crystals, heteropolymers, and the like. Thus, for example, CdSe—CdScore-shell nanocrystals enclosed in a silica shell can be easilyderivatized for coupling to a biological molecule (Bruchez et al. (1998)Science, 281: 2013-2016). Similarly, highly fluorescent quantum dots(e.g., zinc sulfide-capped cadmium selenide) have been covalentlycoupled to biomolecules for use in ultrasensitive biological detection(Warren and Nie (1998) Science, 281: 2016-2018). The use of quantum dotsas labels in the present invention is particularly useful, both indirect sequencing application and more generally as a labelingmethodology for any system set forth herein.

In certain aspects, it is useful to reduce the fluorescence of labelednucleic acids before adding additional labels to the nucleic acids,e.g., to reduce background fluorescence between cycles of labeladdition. This can be accomplished, e.g., by interspersing cycles ofphotobleaching between labeling steps to reduce fluorescence ofpreviously labeled components. For example, nucleic acid templatemolecules are optionally attached to the surface of a microfluidicchannel or to an array member, e.g., a bead or set of beads. A primer isbound to the template, e.g., by flowing the primer across the nucleicacid template. Other sequencing reagents, e.g., a polymerase and aseries of nucleotides are flowed across the template, e.g.,incorporating a nucleotide. For example, a solution comprising at leastone of the four standard nucleotides, at least a portion of whichnucleotides are labeled nucleotides, is flowed across the template. Thechannel is then washed, removing any unincorporated nucleotides. If anucleotide was incorporated, a fluorescence signal associated with theincorporated nucleotide is detected, thus determining the identity ofthe added nucleotide and providing a portion of the sequence. Thetemplate nucleic acid is then photobleached to reduce the backgroundlevel of fluorescence before repeating the procedure with anothernucleotide.

In one preferred sequencing by incorporation method, label is producedindirectly, i.e., incorporation of nucleotide reagents is measured byproduction of a detectable label in a coupled signal reaction, e.g.,when sequencing by pyrophosphate methods.

Polymerase reactions sometimes show incorporation of non-complementarybases at a low frequency, particularly when the bases are present inexcess, or when complementary bases are not present. In one aspect,non-incorporatable nucleotides (e.g., analogues which lack moietieswhich provide for coupling to the phosphate backbone) are added toincrease the fidelity of a polymerase reaction, by competing with thenon-complementary bases. In addition, the non-incorporatable nucleotidesreduce polymerase processivity, which is desirable, e.g., in reactionswhere a complementary residue is not present in the reaction mixture.

In one aspect, chain termination is reversible. See also, Cheeseman U.S.Pat. No. 5,302,509 and Canard U.S. Pat. No. 5,798,210. In one method ofsequencing by synthesis, a “reversible” label is used. In particular, aterminating base comprising a label is added by a polymerase as instandard chain termination methods. The label is cleavable, e.g., byphotolysis, or by exposure to heat or to one or more chemicals, e.g., areducing agent and/or a phosphatase. Base-specific incorporation of thelabel is first detected and then the label is cleaved and washed fromthe array. Alternatively, the label; is cleaved, washed from the arrayand then detected. The array is then exposed to nucleotides comprising alabel and the process is repeated. Sequence information is provided byassessing which nucleotides comprising a label are incorporated andcompiling the information. The typical four nucleotides are optionallyadded in series or they are all added together in one solution. In thelatter case, the four nucleotides each have a detectably differentlabel, which is used to identify the nucleotide incorporated.

Examples of reversible chain terminating nucleotides include, but arenot limited to, nucleotides with 3′-phosphate blocking groups, e.g.,comprising a disulfide, and nucleotides with 3′-carbamate blockinggroups. Example of such compounds include, but are not limited tocompounds having the following formulas:

wherein B comprises a nitrogenous base and R⁴ typically comprises alabel moiety, e.g., a fluorescent label moiety. These nucleotides areadded to a growing nucleotide chain by a polymerase, e.g., taqpolymerase, and then the blocking group is removed to provide a 3′-OHgroup, to which another nucleotide is optionally added as sequencingcontinues.

Removal of the phosphate blocking group comprises reducing the disulfidelinkage with a suitable reducing agent, e.g., including, diborane andother boron-containing reductants, dithiothreitol, and enzymes such asreductases specific for the disulfide group. The cleavage of thedisulfide results in a molecule having the following formula:

which is typically unstable and spontaneously degrades, e.g., throughnucleophilic attack, providing a 3′-phosphate group. The phosphate groupis then cleaved, e.g., using an alkaline phosphatase. Cleavage of the3′-phosphate group leaves an extendable 3′-OH group.

Removal of the carbamate typically comprises reduction of the carbamatelinkage, thus producing an unblocked 3′-OH group, which is optionallyextended, e.g., by addition of another dNTP.

Ribonucleic acid base monomers comprising removable labels having adisulfide linker group at the 3′-hydroxyl group are optionally preparedby normal solid or solution phase phosphoramidite chemistry, optionallyin a microfluidic system. As the 3′-hydroxyl is effectively blocked bythe conjugated linker arm, these monomers can then be used as aterminating residue in standard dideoxy chain termination, or can beused as reversible terminators in sequencing by synthesis protocols asnoted above. The free hydroxy terminus of the linker arm is preferablyderivatized with a detectable label such as a fluorescent orchemiluminescent dye or a radioactive isotope. Methods for preparinglinker arms that can be incorporated into monomers or nucleic acidoligomers are discussed below in the context of nucleic acid oligomers.It is, however, understood that this is by way of example. One of skillwill recognize that a linker that is appropriate for incorporation intoa nucleic acid oligomer synthesis is also optionally utilized toderivatize a nucleic acid monomer.

At least two methods can be utilized to prepare nucleic acids with adisulfide linker having a detectable moiety at one terminus of thelinker. In the first method, the disulfide moiety, preferably suppliedby an agent such as 2-hydroxyethyl disulfide is mono-functionalized atone hydroxy terminus with a detectable group. The remaining hydroxygroup is converted to a phosphoramidite. This linker arm-detectableagent conjugate can then be incorporated into a normal nucleic acidsynthetic cycle.

A second method, exemplified below, allows a trityl protected linker tobe tethered to a growing nucleic acid chain. The trityl group issubsequently removed in a normal nucleic acid deprotection cycle and theliberated hydroxy group is conjugated to another base or to a detectablemoiety.

Synthesis of monotrityl-2-hydroxyethyl disulfide (2)

2-hydroxyethyl disulfide (1, Aldrich Chemical Co.) is monotritylated bythe action of tritychloride and a base such as triethylamine. Thereaction is weighted towards production of the monotrityl derivative byusing a substantial stoichiometric excess (3-5 fold) of 2-hydroxyethyldisulfide relative to tritylchloride. The reaction is typically carriedout in ethyl acetate of a chlorinated hydrocarbon at room temperature.The monotritylated product will exhibit markedly differentchromatographic behavior than both unreacted tritylchloride and theditritylated derivative. Thus, the monotritylated derivative can beeasily purified by, for example, silica gel chromatography, optionallyin a microfluidic system.

Synthesis of monotrityl-2-hydroxyethyldisulfide-2-cyanoethyldiisopropyl-phosphoramidite (3)

The purified monotrityl 2-hydroxyethyl disulfide is converted to aphosphoramidite appropriate for inclusion into solid phase nucleic acidsynthesis as follows. The montrityl compound is contacted with2-cyanoethyldiisopropylchlorophosphoramidite in the presence of anorganic base such as diisopropylethylamine. As the reaction proceeds,amine hydrochloride is produced and precipitates from the reactionmedium. When the reaction is complete, the solid is removed byfiltration and the organic layer is washed with water at approximatelyneutral pH. The solvent is removed by evaporation and the crude productis purified by silica gel chromatography to provide the desired product.

In one embodiment, the phosphoramidite derivative is brought intosolution in, for example, acetonitrile and placed into a reactant vesselon an art-recognized nucleic acid synthesis apparatus. The disulfidelinker arm is added to the growing chain at any desired point in thesynthesis. The use of the phosphoramidite derivative allows thedisulfide linker to be added in a manner identical to any other nucleicacid base phosphoramidite. Following the addition of the disulfidelinker, the trityl group is removed using a standard deprotection cycleand, if desired, chain elongation can then proceed.

In any of the embodiments herein which comprise fixation of targets toarray members, the targets can be related (e.g., sequence fragments of asingle clone) or unrelated. In embodiments where the sequences arerelated, the system optionally includes a microprocessor for compilingoverlapping sequence information.

Direct Sequencing—Real Time Pyrophosphate

One recent approach to sequencing by synthesis is set forth in Ronaghiet al. (1998) “A Sequencing Method Based on Real Time Pyrophosphate”Science 281:363-364 (See also, Nyren and Uhlen (1996) Anal. Biochem.242:84-89 and (1993) 208:171-175 and Canard U.S. Pat. No. 5,798,509). Inthis method, four nucleotides are added stepwise to a template nucleicacid hybridized to a primer. In the applications of the presentinvention, templates are optionally fixed to one or more particlemembers of the arrays. A polymerase adds a nucleotide to the primerbased upon standard base-pairing rules and standard polymerase activity.The addition of a nucleotide to the primer results in the release of aninorganic pyrophosphate from the nucleotide. An ATP sulfurylase enzymeis used to convert the inorganic pyrophosphate into ATP or an ATPanalogue (e.g., comprising a sulfur atom). A luciferase enzyme releaseslight in the presence of the ATP, providing an indication as to when anucleotide is added to the primer. To remove excess ATP from the system,an apyrase is added to degrade the ATP into AMP+2PPi between dNTPaddition cycles. The apyrase also degrades any nucleotide from thesystem which is not added to the primer. Any or all of these reagentscan be present in a reagent train which passes over particle memberscomprising template, or, alternatively can be themselves fixed to arraymembers, where a reagent train comprising the template is passed acrossthe array members. As described above, chemistries for fixing eithernucleic acids or proteins (or both) to any of a variety of array membersis well known. Optionally, the dATP for incorporation into growingnucleic acids is α-thio dATP which can be incorporated by polymerase,but not by luciferase, thus reducing background signal production in theassay.

Indeed, one advantage of the present invention is that it makes thereaction outlined by Ronaghi et al. much more practical. In the Ronaghiet al. reference, due to the use of relatively crude enzyme fractionsand fluidic inefficiencies, signal to noise ratios gradually decreasedas the reaction proceeded (due in part to incomplete washing ofreactants and products between steps), making it difficult to readlonger nucleic acid templates. In contrast, using the present invention,it is possible to isolate completely the relevant reagents, and to washthem from the templates using microfluidic fluid movement.

One additional aspect of the present invention is a new pyrophosphatesequencing reaction. In this new sequencing reaction, PPi is convertedto a thio analogue form of ATP with a sulfurylase. The ATP is combinedwith glucose and converted to glucose-6-phosphate+NADP and ADP.Glucose-6-phosphate dehydrogenase is used to produce NADPH+6phosphoglutamate. The ADP is converted into ATP by addition ofphosphoenoyl pyruvate (PEP) and a phosphokinase enzyme. ATP formationcan be monitored with luciferase. Preferably, NADPH formation can bemonitored by monitoring fluorescence. In essence, as long as PEP ispresent in the reaction, this set of reactions provides a signalamplification cycle by producing additional ATPs. This is helpful inluciferase mediated signals, because luciferase produces a relativelylow level of detectable signal, e.g., as compared to fluorescence.

Maxam Gilbert Sequencing

In addition to chain termination and coupled enzymatic methods, chemicalnucleic acid degradation methods have been in use for specializedapplications; see, Maxam and Gilbert (1980) in Grossman and Moldave(eds.) Academic Press, New York, Methods in Enzymology 65:499-560. Aswith the sequencing methodologies noted above, the Maxam Gilbert methodis entirely amenable to use with the arrays of the invention. Inparticular, the template can be fixed to an array (covalently ornon-covalently) and selectively degraded by the Maxam Gilbertdegradation method. Reaction products can be viewed concurrent withdegradation, or following degradation (i.e., downstream of thereaction). Indeed, one advantage of the present invention over standardMaxam Gilbert methods is that extremely small quantities of reagent canbe used. One of skill will appreciate that some of the reagents in theMaxam Gilbert method are highly toxic and/or explosive, making workingwith large quantities of these reagents (i.e., as in standard methods)somewhat problematic.

Sequencing by Hybridization

Sequencing by hybridization to complementary oligonucleotides has alsobeen developed e.g., in U.S. Pat. No. 5,202,231, to Drmanac et al.;Drmanac et al. (1989) Genomics 4:114-128 and, e.g., Drmanac et al.(1998) “Accurate sequencing by hybridization for DNA diagnostics andindividual genomics” Nature Biotechnology 16: 54-58. Methods ofdetecting genetic differences by hybridization are described e.g., inFodor (1997) “Genes, Chips and the Human Genome” FASEB Journal.11:121-121; Fodor (1997) “Massively Parallel Genomics” Science.277:393-395; Chee et al. (1996) “Accessing Genetic Information withHigh-Density DNA Arrays” Science 274:610-614, and in a variety of otherpublications.

The arrays of the present invention are particularly well suited tosequencing and/or detection of differences at the nucleic acid level, byhybridization methods. In particular, either probe or target nucleicacids (or both in multiplexed assays) are fixed to array members. Eitherprobes, or target members, or both, are flowed sequentially,simultaneously or in parallel into contact. Typically, either the probeor target member are labeled to facilitate detection of anyhybridization event. Alternatively, nucleic acids can simply behybridized to and denatured from the array, with detection occurringdownstream from the hybridization event (e.g., where the detection andhybridization are timed to provide meaningful information regarding thehybridization). Downstream detection can be performed with a labeledprobe (which is optionally a component of the array, or of a secondfluidly connected array), or by simple detection of the physicalpresence of the nucleic acid (e.g., detection downstream can beperformed by mass spectroscopy).

It is expected that one of skill is thoroughly familiar with the theoryand practice of nucleic acid hybridization and probe selection. Gait,ed. Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford(1984); W. H. A. Kuijpers Nucleic Acids Research 18(17), 5197 (1994); K.L. Dueholm J. Org. Chem. 59, 5767-5773 (1994); S. Agrawal (ed.) Methodsin Molecular Biology, volume 20; and Tijssen (1993) LaboratoryTechniques in biochemistry and molecular biology—hybridization withnucleic acid probes, e.g., part I chapter 2 “overview of principles ofhybridization and the strategy of nucleic acid probe assays”, Elsevier,New York provide a basic guide to nucleic acid hybridization.Hybridization of nucleic acids to nucleic acids fixed to solidsubstrates is described, e.g., in U.S. Pat. No. 5,202,231, to Drmanac etal.; Drmanac et al. (1989) Genomics 4:114-128 and, e.g., Drmanac et al.(1998) “Accurate sequencing by hybridization for DNA diagnostics andindividual genomics” Nature Biotechnology 16: 54-58. Methods ofdetecting genetic differences by hybridization are described e.g., inFodor (1997) “Genes, Chips and the Human Genome” FASEB Journal.11:121-121; Fodor (1997) “Massively Parallel Genomics” Science.277:393-395; Chee et al. (1996) “Accessing Genetic Information withHigh-Density DNA Arrays” Science 274:610-614.

These methods of sequencing and assessing genetic difference are adaptedto the arrays of the invention by performing hybridization assays in amicrofluidic format. In particular, nucleic acids (e.g. either probes ortargets) are fixed to an array member e.g., by covalent synthesisstrategies as noted supra (e.g. by light directed synthesis andphotomasking procedures), or by hybridization to a molecule whichcaptures the nucleic acid, such as a complementary nucleic acid, orantibody specific for DNA or RNA. In addition to the references notedabove regarding nucleic acid hybridization, antibodies to, e.g., DNA,RNA and DNA-RNA duplexes are known, as are immunological methods ofscreening for (and differentiating between) DNA, RNA and RNA-DNA. Forexample, Coutlee et al. (1989) Analytical Biochemistry 181:153-162describe non-isotopic detection of RNA in an enzyme immunoassay using amonoclonal antibody which binds DNA:RNA hybrids. In these assays,hybridization of an RNA target with a biotinylated DNA probe isperformed, followed by incubation of the hybridized target-probe duplexon an anti-biotin plate, reaction of the resulting bound duplex with abeta-galactosidase labeled monoclonal antibody specific for RNA-DNAhybrids, and addition of a fluorescent substrate. In another example, a“sandwich” hybridization method is described for non-isotopic detectionof e.g., RNA using oligonucleotides (Ishii & Ghosh (1993) BioconjugateChem. 4:34-41). In these assays, the RNA target is hybridized to a firstcomplementary oligonucleotide, which is linked to a bead. The RNA targetis then hybridized to a second complementary oligonucleotide conjugatedto alkaline phosphatase. The RNA target is detected by providing achemiluminescent alkaline phosphatase substrate. These methods arereadily adapted to the present invention, e.g., by providing arrays ofsuch beads in a microfluidic format, as well as downstream, sequential,or simultaneous detection of the alkaline phosphatase reaction.

For example, in one aspect, targets for sequencing are fixed toparticles (for example, unsequenced clones can be fixed to particlesets, or specific fragments of such clones can be fixed to particularparticle sets. The particle sets are then flowed into e.g., selectedportions of a particle retention region. Small labeled probes (e.g.,6-15 mers, typically 6-12 mers) are then flowed into contact with thefixed targets for sequencing under selected hybridization conditions(typically stringent hybridization conditions), and the arrays aremonitored for specific binding by the probes. The probes are then washedfree of the targets and the process repeated with at least oneadditional probe. Specific sequences are generated by compiling thesequences of probes bound to the targets. Examples of devices forstoring and accessing large sets of small probes in conjunction with amicrofluidic system are described, e.g., in “Closed Loop BiochemicalAnalyzers” WO 98/45481.

Other investigators have also reported immunological detection ofDNA:RNA hybrids, including Bogulayski et al. (1986) J. Immunol. Methods89:123-130; Prooijen-Knegt (1982) Exp. Cell Res. 141:397-407; Rudkin(1976) Nature 265:472-473, and Stollar (1970) PNAS 65:993-1000.Similarly, detection of DNA:DNA hybrids and RNA:RNA hybrids has alsobeen described. See, Ballard (1982) Mol. Immunol. 19:793-799; Pisetskyand Caster (1982) Mol. Immunol. 19:645-650, and Stollar (1970) PNAS65:993-1000. These methods are similarly adapted to the presentinvention by fixing one or more component of the assay to one or morearray members, flowing other components of the assay into contact withthe one or more array members and detecting any resulting signal.

An alternative sequencing method useful in the present invention, e.g.,using particle arrays and microfluidic devices, involves the use of anintercalator. A template is sequenced in the presence of anintercalator, e.g., an intercalating nucleic acid dye. Nucleotides areflowed across the template or the template is flowed across a nucleotidesolution as described above. Upon addition of a nucleotide to theprimer, the intercalator, e.g., a fluorescent or chemiluminescentintercalator, intercalates into the new double stranded region producedby the addition of a nucleotide to the primer. The intercalator is thendetected, e.g., by an increase in signal, i.e., a fluorescent signal.The signal is optionally photobleached after detection, as describedabove, to decrease the background signal before addition of morenucleotides.

The above sequencing methods are optionally performed using themicrofluidic devices and particle arrays of the invention to provide,e.g., a high throughput system of sequencing. A schematic of such asystem is provided in FIG. 17. FIG. 17 shows three 384-well microtiterplates, plates 1705, 1710, and 1715. Each well contains a set ofparticles comprising a nucleic acid template. Therefore, the systemshown optionally comprises 1152 different nucleic acid templates thatare optionally sequenced in a high throughput manner. Additionalmicrowell plates and channels are optionally used to provide a greaternumber of templates. A plate of blank particle sets is also optionallyincluded, e.g., plate 1720. The particle sets are loaded into a set ofcapillaries or channels as shown by capillary set 1725. For example, 96particle sets are optionally loaded into each of 12 channels using 12sipper capillaries or one sipper capillary fluidly coupled to each ofthe 12 channels. The particle sets are typically retained in thecapillary or microchannel by a porous particle retention element, e.g.,a sintered glass frit, a set of epoxy coated particles, or the like.Alternative particle retention devices are described above, e.g.,narrowed channel dimensions. The particle retention element fixes orretains the particle sets, e.g., particle sets comprising nucleic acidtemplates, in the channel. The particle sets, e.g., templates, are thenoptionally exposed to a series or train of reagents. The reagents aretypically added through each capillary, e.g., from another set ofmicrowell plates, to perform various assays, e.g., sequencing. A singlecontroller, e.g., controller 1730 is optionally used to control fluidflow through the sipper and channels. One or more detector is used tomonitor the particle packets in the channels as various nucleotides areadded. Alternatively, detectors are positioned downstream of thechannels to monitor the waste products, e.g., to detect a fluorescentlabel that has since been washed from the channels. For exampledetection optionally occurs in detection region 1735. Using a systemsuch as that shown in FIG. 17, one particle set is optionally loaded inabout one minute. Therefore 96 templates are optionally analyzed, e.g.,sequenced, in 1.6 hours. Alternatively, particles with differentchemistries are arrayed sequentially in a single capillary and atemplate is flowed across the array, e.g., for sequencing.

In addition to sequencing by hybridization, essentially similar methodscan be used for determination of genetic difference, assays fordetermining nucleic acid melting points, and the like. Additionaldetails on these procedures are found herein.

PCR

In addition to its applicability to sequencing, PCR is desirablypracticed using the arrays of the invention. In particular, PCRtemplates and/or reagents (e.g., a thermostable polymerase) can be fixedto particles, as described herein and using techniques available in theart. Reagents and/or templates can be passed over arrays, where PCR isperformed. This format is especially useful where several PCR productsare to be screened simultaneously (or sequentially). A variety of suchPCR assays, e.g., for diagnostic applications (e.g., detection ofviruses such as HIV, HBV, HCV, etc., detection of infectious organisms(bacteria, parasites, etc.), detection of genetic abnormalities (geneticdiseases, cancer, etc.), as well as for research applications (e.g.,screening of drugs, drug targets, genes effected in vivo or in vitro bydrugs or potential drugs, results of forced evolution methods) as wellas many others are well known in the literature and adaptable to thepresent invention.

Examples of techniques sufficient to direct persons of skill through invitro amplification methods, including the polymerase chain reaction(PCR) the ligase chain reaction (LCR), Q-replicase amplification andother RNA polymerase mediated techniques (e.g., NASBA) are found inBerger, Sambrook, and Ausubel, as well as Mullis et al., (1987) U.S.Pat. No. 4,683,202; PCR Protocols A Guide to Methods and Applications(Innis et al. eds) Academic Press Inc. San Diego, Calif. (1990) (Innis);Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIHResearch (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA86, 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874;Lomell et al. (1989) J. Clin. Chem. 35, 1826; Landegren et al., (1988)Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8, 291-294; Wuand Wallace, (1989) Gene 4, 560; Barringer et al. (1990) Gene 89, 117,and Sooknanan and Malek (1995) Biotechnology 13: 563-564. Improvedmethods of cloning in vitro amplified nucleic acids are described inWallace et al., U.S. Pat. No. 5,426,039. Improved methods of amplifyinglarge nucleic acids by PCR are summarized in Cheng et al. (1994) Nature369: 684-685 and the references therein, in which PCR amplicons of up to40 kb are generated. One of skill will appreciate that essentially anyRNA can be converted into a double stranded DNA suitable for restrictiondigestion, PCR expansion and sequencing using reverse transcriptase anda polymerase. See, Ausubel, Sambrook and Berger, all supra.

It will be appreciated that these benchtop uses for PCR are adaptable tomicrofluidic systems. Indeed, PCR amplification is particularly wellsuited to use in the apparatus, methods and systems of the invention.

Thermocycling amplification methods, including PCR and LCR, areconveniently performed in microscale devices, making iterative fluidicoperations involving PCR well suited to use in methods and devices ofthe present invention (see also, U.S. Pat. Nos. 5,498,392 and 5,587,128to Willingham et al.).

Thermocycling for PCR and other thermocyclic applications (e.g., theligase chain reaction, or LCR) can be conducted in microfluidic systemsin at least two ways. First, a heat source (external or internal) can beused to thermocycle all or part of a device, thereby heating and coolingthe array within the microfluidic system. In a second approach, jouleheating is used. Thermocycling in microscale devices, e.g., using jouleheating, is described in co-pending application Ser. No. 08/977,528,filed Nov. 25, 1997 (now U.S. Pat. No. 5,965,410). In brief, energy isprovided to heat fluids, e.g., samples, analytes, buffers and reagents,in desired locations of the substrates in an efficient manner byapplication of electric current to fluids in microchannels. Thus, thepresent invention optionally uses power sources that pass electricalcurrent through the fluid in a channel for heating purposes, as well asfor material transport. In exemplary embodiments, the fluid passesthrough a channel of a desired cross-section (e.g., diameter) to enhancethermal transfer of energy from the current to the fluid. The channelscan be formed on almost any type of substrate material such as, forexample, amorphous materials (e.g., glass, plastic, silicon),composites, multi-layered materials, combinations thereof, and the like.In general, electric current passing through the fluid in a channelproduces heat by dissipating energy through the electrical resistance ofthe fluid. Power dissipates as the current passes through the fluid andgoes into the fluid as energy as a function of time to heat the fluid.The following mathematical expression generally describes a relationshipbetween power, electrical current, and fluid resistance, i.e., POWER=I²Rwhere POWER=power dissipated in fluid; I=electric current passingthrough fluid; and R=electric resistance of fluid.

The above equation provides a relationship between power dissipated(“POWER”) to current (“I”) and resistance (“R”). In some of theembodiments, which are directed toward moving fluid in channels, e.g.,to provide mixing, electrophoretic separation, or the like, a portion ofthe power goes into kinetic energy of moving the fluid through thechannel. However, it is also possible to use a selected portion of thepower to controllably heat fluid in a channel or selected channelregions. A channel region suitable for heating is often narrower orsmaller in cross-section than other channel regions in the channelstructure, as a smaller cross-section provides higher resistance in thefluid, which increases the temperature of the fluid as electric currentpasses through. Alternatively, the electric current is increased acrossthe length of the channel by increased voltage, which also increases theamount of power dissipated into the fluid to correspondingly increasefluid temperature.

The introduction of electrical current into fluid causes heat (Jouleheating). In the examples of fluid movement herein where thermal effectsare not desired, the heating effect is minimal because, at the smallcurrents employed, heat is rapidly dissipated into the chip itself. Bysubstantially increasing the current across the channel, rapidtemperature changes are induced that can be monitored by conductivity.At the same time, the fluid can be kept static in the channel by usingalternating instead of direct current. Because nanoliter volumes offluid have tiny thermal mass, transitions between temperatures can beextremely short. Oscillations between any two temperatures above 0° C.and below 100° C. in 100 milliseconds have been performed. Additionalapplications of joule heating to sequencing methodologies is set forthin “Closed Loop Biochemical Analyzers” (WO 98/45481).

Melting Point Analysis of Nucleic Acids

In an embodiment similar to sequencing by hybridization, the systems,devices arrays and methods of the present invention can be used todetect variations in nucleic acid sequences by determining the strengthof the hybridization between the targeted nucleic acid and probes thatare putative perfect complements to the target. By identifying thedifference in stability between the imperfect and perfect hybrids underconditions of increasing hydrogen bond stress, one can identify thosenucleic acids that contain a variation.

In practice, a microfluidic device is configured to accept a samplecontaining an amplified nucleic acid or polynucleotide sequence ofinterest, convert it to single-stranded form, facilitate hybridizationwith a nucleic acid probe, such as an oligonucleotide, and then subjectthe hybridization mixture to a chemical or temperature gradient thatdistinguishes between perfectly matched targets and those that differ byat least one base pair (mismatch). Either the probe or the template canbe fixed to an array component. In some embodiments, one or more loci ortargeted areas of the sample polynucleotide are first amplified bytechniques such as PCR or sandwich hybridization. In other embodiments,unamplified polynucleotide is provided to the device and amplifiedtherein.

Hybridization of the probe results in a perfect hybrid with nomismatches when the sample polynucleotide contains the complementarysequence, i.e., no variation, or in a hybrid with mismatches if thesample polynucleotide differs from the probe, i.e., contains a sequencevariation. The stability of the imperfect hybrid differs from theperfect hybrid under conditions of increasing hydrogen bond stress. Avariety of methods are available for subjecting the hybrids toincreasing hydrogen bond stress, sufficient to distinguish betweenperfectly matched probe/target hybrids and imperfect matches. Forexample, the hybrids are optionally subjected to a temperature gradient,or alternatively, can be subjected to increasing concentrations of achemical denaturant, e.g., formamide, urea, and the like, or increasingpH. By monitoring hybridization between one or more array component andone or more unknown nucleic acid, it is possible to determine percentsequence complementarity.

The assay is optionally repeated several times, varying theconcentration of denaturant or temperature with each successive assay.By monitoring the level of hybridization, one can determine theconcentration of denaturant at which the probe-target hybrid isdenatured. This level is then compared to a standard curve, to determinewhether one or more variations are present in the nucleic acid.

Other Sequencing Strategies

Other sequencing methods which reduce the number of steps necessary fortemplate preparation and primer selection have been developed. Oneproposed variation on sequencing technology involves the use of modularprimers for use in PCR and DNA sequencing. For example, Ulanovsky andco-workers have described the mechanism of the modular primer effect(Beskin et al. (1995) Nucleic Acids Research 23(15):2881-2885) in whichshort primers of 5-6 nucleotides can specifically prime atemplate-dependent polymerase enzyme for template dependent nucleic acidsynthesis. A modified version of the use of the modular primer strategy,in which small nucleotide primers are specifically elongated for use inPCR to amplify and sequence template nucleic acids has also beendescribed. The procedure is referred to as DNA sequencing usingdifferential extension with nucleotide subsets (DENS). See, Raja et al.(1997) Nucleic Acids Research 25(4):800-805. These modular primerstrategies, for sequencing or PCR, are readily adapted to the presentmethods and arrays. For example, template or primer nucleic acids can befixed, directly or indirectly, to the array members of the presentinvention. The corresponding template or primer is flowed into contactwith the array member, along with any other components of the sequencingor PCR reaction (or other reaction, if appropriate) and the reactionperformed under appropriate conditions to the reaction. Products aredetected e.g., by washing the products from the array and detecting theproducts at a downstream detector.

Liquid Crystal Assay Systems

In still another embodiment, binding of a protein to a target componentsuch as a nucleic acid can be detected by the use of liquid crystals.Liquid crystals have been used, for example, to amplify and transducereceptor-mediated binding of proteins at surfaces into optical outputs.Spontaneously organized surfaces can be designed so that a protein, uponbinding to a nucleic acid hosted on the surface of an array memberherein, triggers changes in the orientations of 1- to20-micrometer-thick films of supported liquid crystals, thuscorresponding to a reorientation of ˜10⁵ to 10⁶ mesogens per protein.Binding-induced changes in the intensity of light transmitted throughthe liquid crystal are easily seen with the naked eye and can be furtheramplified by using surfaces designed so that protein-nucleic acidbinding causes twisted nematic liquid crystals to untwist (see, e.g.,Gupta et al. (1998) Science, 279: 2077-2080). This approach to thedetection of protein/nucleic acid interactions does not require labelingof the analyte, does not require the use of electroanalytical apparatus,provides a spatial resolution of micrometers, and is sufficiently simplethat it is useful in biochemical assays and imaging of spatiallyresolved chemical libraries.

Diagnostic/Screening Assays

In one aspect of the present invention, diagnostic assays are provided.As discussed supra, assays can take the form of nucleic acid detectionor sequencing assays which screen for the presence or absence or type ofa nucleic acid. The presence or type of a nucleic acid in a biologicalsample is an indicator for the presence of, e.g., an infectious organism(e.g., virus, bacteria, fungal cell or the like) in the biologicalsample. Thus, detection of a nucleic acid provides an indication that,e.g., a patient is infected with such an infectious organism.

Similarly, the presence of certain mRNAs (e.g., mRNAs from oncogeneproducts) and/or specific sequences in genomic DNA are correlated with avariety of disease states, including, e.g., cancer. Thus, any of thevarious assay formats described herein can be used for the detection ofspecific nucleic acids which correspond to particular disease states.Many such correlations are well established in the art.

Microfluidic devices are very efficient at capturing molecules in afluid stream, e.g., in functionalized channels, or on particle setswithin the channels. After capturing a particular molecule, e.g., asample that is present only in small concentrations, the devices areused to detect the molecule, e.g., its presence or type. For example, anumber of nucleic acids or cells are optionally flowed through amicrofluidic device for capture, e.g., by a set of particles. Reagentsthat bind to a molecule of interest, e.g., by hybridization, areoptionally flowed across the captured molecules and a molecule ofinterest is identified, e.g., by its binding specificity. This isespecially useful when samples of low concentration are captured becausethe sample is optionally positioned in a defined location for furtheranalysis or detection, e.g., by specific binding moieties.

In addition to screening for diseases, the arrays of the invention canalso be used to select for the presence of desirable traits. Forexample, in agriculture, many correlations between desirable traits andparticular genomic or RNA sequences are well established. For example,crops such as corn, soybean, cotton, potatoes, tomatoes, wheat, millet,and many others are routinely selected, in part, by selecting for thepresence or absence of nucleic acids which are correlated with desirabletraits such as yield, disease resistance, herbicide resistance, droughttolerance and the like.

Expression Profiling

One particularly useful aspect of array technology is the ability toprofile expression of one or more expression products for one or morebiological sample. By profiling expression of RNAs and/or proteins, itis possible to determine whether certain disease-related genes (e.g.,oncogenes, infectious organisms, or the like) are expressed. Inaddition, it is possible to use expression profiles to determinecombinatorial genetic and polygenetic effects on expression and topredict phenotypes resulting from polygenetic effects. For example,where expression of several genes causes a phenotype, it is possible tomonitor expression of these genes on the arrays of the invention. Forexample, polygenic effects often underlie disease or herbicideresistance in plants, pesticide resistance in insects, and diseasestates in animals (including humans).

A variety of sources of biological material can be profiled, includinganimal sources (including human, vertebrate, mammalian, insect, etc.)and plant sources (e.g., wild or domesticated plants, such as cropplants). The biological sources can be from tissues, cells, wholeorganisms, cell cultures, tissue cultures, or the like. A variety ofprofiling methods are adaptable to the present system, includinghybridization of expressed or amplified nucleic acids to a nucleic acidarray, hybridization of expressed polypeptides to a protein array,hybridization of peptides or nucleic acids to an antibody array,subtractive hybridization, differential display and others.

In one preferred embodiment, the expression products which are detectedin the methods of the invention are RNAs, e.g., mRNAs expressed fromgenes within a cell derived from the biological source to be profiled. Anumber of techniques are available for detecting RNAs, which can beutilized or adapted to the arrays of the invention. For example,northern blot hybridization is widely used for RNA detection, and isgenerally taught in a variety of standard texts on molecular biology,including: Berger and Kimmel, Sambrook, Ausubel (all supra), etc.Furthermore, one of skill will appreciate that essentially any RNA canbe converted into a double stranded DNA using a reverse transcriptaseenzyme and a polymerase. See, Ausubel, Sambrook and Berger, id. Thus,detection of mRNAs can be performed by converting, e.g., mRNAs intoDNAs, which are subsequently detected in, e.g., a “Southern blot”format.

Furthermore, DNAs can be amplified to aid in the detection of raremolecules by any of a number of well known techniques, including: thepolymerase chain reaction (PCR), the ligase chain reaction (LCR),Q-replicase amplification and other RNA polymerase mediated techniques(e.g., NASBA). Examples of these techniques are found in Berger,Sambrook, and Ausubel, id., as well as in those references noted supraregarding in vitro amplification. These amplification steps can beperformed in the microfluidic system, or external to the microfluidicsystem.

Probes can be fixed to array members to create a probe array, andexpression products (or in vitro amplified nucleic acids correspondingto expression products) can be labeled and hybridized with the array.For convenience, it may be helpful to use several arrays simultaneously(e.g., in the same or in separate microfluidic devices), or to usearrays of large or small numbers of members, depending on the number ofexpression products to be detected.

It will be appreciated that probe design is influenced by the intendedapplication. For example, where several allele-specific probe-targetinteractions are to be detected in a single assay, e.g., on a singlearray, it can be desirable to have similar melting temperatures for allof the probes (or course this is not necessary, as joule or zone heatingcan be used to maintain different portions of an array at differenttemperatures). Accordingly, the length of the probes are optionallyadjusted so that the melting temperatures for all of the probes on thearray are closely similar (it will be appreciated that different lengthsfor different probes may be needed to achieve a particular T_(m) wheredifferent probes have different GC contents). Although meltingtemperature is a primary consideration in probe design, other factorsare also optionally used to further adjust probe construction, such aselimination of self-complementarity in the probe (which can inhibithybridization of a target nucleotide).

One way to compare expression products between two cell populations isto identify mRNA species which are differentially expressed between thecell populations (i.e., present at different abundances between the cellpopulations). In addition to the techniques noted above, anotherpreferred method is to use subtractive hybridization (Lee et al. (1991)Proc. Natl. Acad. Sci. (U.S.A.) 88:2825) or differential displayemploying arbitrary primer polymerase chain reaction (PCR) (Liang andPardee (1992) Science 257:967). Each of these methods has been used byvarious investigators to identify differentially expressed mRNA species.See, Salesiotis et al. (1995) Cancer Lett. 91:47; Jiang et al. (1995)Oncogene 10:1855; Blok et al. (1995) Prostate 26:213; Shinoura et al.(1995) Cancer Lett. 89:215; Murphy et al. (1993) Cell Growth Differ4:715; Austruy et al. (1993) Cancer Res. 53:2888; Zhang et al. (1993)Mol. Carcinog. 8:123; and Liang et al. (1992) Cancer Res. 52:6966). Themethods have also been used to identify mRNA species which are inducedor repressed, e.g., by drugs or certain nutrients (Fisicaro et al.(1995) Mol. Immunol. 32:565; Chapman et al. (1995) Mol. Cell.Endocrinol. 108:108; Douglass et al. (1995) J. Neurosci. 15:2471; Aielloet al. (1994) Proc. Natl. Acad. Sci. (U.S.A.) 91:6231; Ace et al. (1994)Endocrinology 134:1305.

For the technique of differential display, Liang and Pardee (1992),supra provide theoretical calculations for the selection of 5′ and 3′arbitrary primers. Correlation of observed results to the theory is alsoprovided. In practice, 5′ primers of less than about 9 nucleotides maynot provide adequate specificity (slightly shorter primers of about 8 to10 nucleotides have been used in PCR methods for analysis of DNApolymorphisms. See also, Williams et al. (1991) Nucleic Acids Research18, 6531). The primer(s) optionally comprise 5′-terminal sequences whichserve to anchor other PCR primers (distal primers) and/or which comprisea restriction site or half-site or other ligatable end. Where arestriction site or amplification template for a second primer isincorporated, the primers are optionally longer than those describedabove by the length of the restriction site, or amplification templatesite. Standard restriction enzyme sites include 4 base sites, 5 basesites, 6 base sites, 7 base sites, and 8 base sites. An amplificationtemplate site for a second primer can be of essentially any length, forexample, the site can be about 15-25 nucleotides in length. Any ofprimers, templates, or other reactants (e.g., enzymes) can be fixed toarray particle members.

The amplified products are optionally labeled and are typically resolvede.g., by electrophoresis on a polyacrylamide gel or other sieving matrixin the microfluidic system; the location(s) where label is present arerecovered from the sieving matrix, typically by elution orelectrokinetic methods. The resultant recovered product species can besubcloned into a replicable vector with or without attachment oflinkers, amplified further, and/or detected, or even sequenced directly.As noted, direct sequencing of PCR generated amplicons by selectivelyincorporating boronated nuclease resistant nucleotides into theamplicons during PCR and digestion of the amplicons with a nuclease toproduce sized template fragments has been performed (Porter et al.(1997) Nucleic Acids Research 25(8):1611-1617) and is applicable to thepresent invention.

It is expected that one of skill can use, e.g., differential display forexpression profiling. In addition, companies such as CuraGen Corp. (NewHaven Conn.) provide robust expression profiling based upon differentialdisplay techniques. See, e.g., WO 97/15690 by Rothenberg et al., andthese methods are readily adapted to a microfluidic format.

Expression Profiling of Proteins

In addition to nucleic acid formats, the arrays of the invention caneasily be adapted to screening other biological components as well,including cells, antibodies, antibody ligands and the like. The presenceor absence of such cells, antibodies and antibody ligands are also knownto correlate with desirable or undesirable features. For example, onecommon assay for the detection of infectious organisms involves an ELISAassay or western blot to detect the presence of antibodies in a patientto a particular infectious agent (e.g., an HIV virus). Suchimmunological assays are also adaptable to the arrays of the presentinvention, e.g., by fixing an antibody or antibody target to an arraymember and exposing the array member to the corresponding antibody orantibody target. Thus, immunological reagents (i.e., those used in anassay in which an antibody is a target or reagent) can be flowed acrossthe arrays of the invention.

In addition to profiling RNAs (or corresponding cDNAs) as describedabove, it is also possible to profile proteins. In particular, variousstrategies are available for detecting many proteins simultaneously. Asapplied to the present invention, detected proteins, corresponding toexpression products, can be derived from one of at least two sources.First, the proteins which are detected can be either directly isolatedfrom a cell or tissue to be profiled, providing direct detection (and,optionally, quantification) of proteins present in a cell. Second, mRNAscan be translated into cDNA sequences, cloned and expressed. Thisincreases the ability to detect rare RNAs, and makes it possible toimmediately associate a detected protein with its coding sequence. Forpurposes of the present invention, even an out of frame peptide is anindicator for the presence of a corresponding RNA.

A variety of hybridization techniques, including western blotting, ELISAassays, and the like are available for detection of specific proteins.See, Ausubel, Sambrook and Berger, supra. See also, Antibodies: ALaboratory Manual, (1988) E. Harlow and D. Lane, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. Non-hybridization based techniquessuch as two-dimensional electrophoresis can also be used tosimultaneously and specifically detect large numbers of proteins. Eitherantibody or two dimensional gel electrophoresis can readily be adaptedto microfluidic systems.

One typical technology for detecting specific proteins involves makingantibodies to the proteins. By specifically detecting binding of anantibody and a given protein, the presence of the protein can bedetected. In addition to available antibodies, one of skill can easilymake antibodies using existing techniques, or modify those antibodieswhich are commercially or publicly available. In addition to the artreferenced above, general methods of producing polyclonal and monoclonalantibodies are known to those of skill in the art. See, e.g., Paul (ed.)(1993) Fundamental Immunology, Third Edition Raven Press, Ltd., New YorkColigan (1991) Current Protocols in ImmunologyWiley/Greene, N.Y.; Harlowand Lane (1989) Antibodies: A Laboratory Manual Cold Spring HarborPress, NY; Stites et al. (eds.) Basic and Clinical Immunology (4th ed.)Lange Medical Publications, Los Altos, Calif., and references citedtherein; Goding (1986) Monoclonal Antibodies: Principles and Practice(2d ed.) Academic Press, New York, N.Y.; and Kohler and Milstein (1975)Nature 256:495-497. Other suitable techniques for antibody preparationinclude selection of libraries of recombinant antibodies in phage orsimilar vectors. See, Huse et al. (1989) Science 246:1275-1281; and Wardet al. (1989) Nature 341:544-546. Specific monoclonal and polyclonalantibodies and antisera will usually bind with a KD of at least about0.1 μM, preferably at least about 0.01 μM or better, and most typicallyand preferably, 0.001 μM or better. As used herein, an “antibody” refersto a protein consisting of one or more polypeptide substantially orpartially encoded by immunoglobulin genes or fragments of immunoglobulingenes. The recognized immunoglobulin genes include the kappa, lambda,alpha, gamma, delta, epsilon and mu constant region genes, as well asmyriad immunoglobulin variable region genes. Light chains are classifiedas either kappa or lambda. Heavy chains are classified as gamma, mu,alpha, delta, or epsilon, which in turn define the immunoglobulinclasses, IgG, IgM, IgA, IgD and IgE, respectively. A typicalimmunoglobulin (antibody) structural unit is known to comprise atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (VH) refer to these light and heavy chainsrespectively. Antibodies exist as intact immunoglobulins or as a numberof well characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′2, a dimer ofFab which itself is a light chain joined to VH—CH1 by a disulfide bond.The F(ab)′2 may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)₂ dimer into anFab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1993), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Antibodies include single chainantibodies, including single chain Fv (sFv) antibodies in which avariable heavy and a variable light chain are joined together (directlyor through a peptide linker) to form a continuous polypeptide.

For purposes of the present invention, antibodies or antibody fragmentscan be arrayed, e.g., by coupling to an amine moiety fixed to a solidphase particle array member, in a manner similar to that described abovefor construction of nucleic acid arrays. As above for nucleic acidprobes, the antibodies can be labeled, or proteins corresponding toexpression products can be labeled. In this manner, it is possible tocouple hundreds, or even thousands, of different antibodies to membersof an array. In one embodiment, a bacteriophage antibody display libraryis screened with a polypeptide encoded by a cell, or obtained byexpression of mRNAs, differential display, subtractive hybridization orthe like. Combinatorial libraries of antibodies have been generated inbacteriophage lambda expression systems which are screened asbacteriophage plaques or as colonies of lysogens (Huse et al. (1989)Science 246:1275; Caton and Koprowski (1990) Proc. Natl. Acad. Sci.(U.S.A.) 87:6450; Mullinax et al (1990) Proc. Natl. Acad. Sci. (U.S.A.)87:8095; Persson et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88:2432).Various embodiments of bacteriophage antibody display libraries andlambda phage expression libraries have been described (Kang et al.(1991) Proc. Natl. Acad. Sci. (U.S.A.) 88:4363; Clackson et al. (1991)Nature 352:624; McCafferty et al. (1990) Nature 348:552; Burton et al.(1991) Proc. Natl. Acad. Sci. (U.S.A.) 88:10134; Hoogenboom et al.(1991) Nucleic Acids Res. 19:4133; Chang et al. (1991) J. Immunol.147:3610; Breitling et al. (1991) Gene 104:147; Marks et al. (1991) J.Mol. Biol. 222:581; Barbas et al. (1992) Proc. Natl. Acad. Sci. (U.S.A.)89:4457; Hawkins and Winter (1992) J. Immunol. 22:867; Marks et al.(1992) Biotechnology 10:779; Marks et al. (1992) J. Biol. Chem.267:16007; Lowman et al (1991) Biochemistry 30:10832; Lerner et al.(1992) Science 258:1313.

The patterns of hybridization which are detected on the array provide anindication of the presence or absence of expressed protein sequences. Aslong as the library or array against which a population of proteins areto be screened can be correlated from one experiment to the next (e.g.,by noting the x-y coordinates of the library or array member, or bynoting the position of markers within the arrays (e.g., where the arrayscomprise mobile members)), no sequence information is required tocompare expression profiles from one representative sample to another.In particular, the mere presence or absence (or degree) of labelprovides the ability to determine differences. One advantage of usingarrayed libraries of antibodies for protein detection is that theindividual library members can be uncharacterized.

More generally, peptide and nucleic acid hybridization to arrays orlibraries (or even simple two dimensional gels) can be treated in amanner analogous to a bar code label. Any diverse library or array canbe used to screen for the presence or absence of complementarymolecules, whether RNA, DNA, protein, or a combination thereof. Bymeasuring corresponding signal information between different sources oftest material (e.g., different hybrid or inbred plants, or differenttissues, or the like), it is possible to determine differences inexpression products for the different source materials. As set forthbelow, this process is facilitated by various high throughput integratedsystems set forth below.

In addition to array based approaches, mass spectrometry is in use foridentification of large sets of proteins in samples, and is suitable foridentification of many proteins in a sequential or parallel fashion. Forexample, Hutchens et al. U.S. Pat. No. 5,719,060, describe methods andapparatus for desorption and ionization of analytes for subsequentanalysis by mass spectroscopy and/or biosensors. In the presentinvention, components can be released from array members in a sequentialfashion and prepared for mass spectrometry.

Two and three dimensional gel based approaches can also be used for thespecific and simultaneous identification and quantification of largenumbers of proteins from biological samples. Multi-dimensional geltechnology is well-known and described e.g., in Ausubel, supra, Volume2, Chapter 10. As applied to microfluidic systems, intersecting channelscan comprise different separation media. After flowing componentsthrough a first media, components can be flowed through a second mediain a second channel. The components can be labeled, e.g., by flowingstaining reagents into contact with the components. The labeledcomponents are then flowed past a suitable detector (or the entiremicrofluidic system can be imaged simultaneously, e.g., using a CCDarray). Image analysis of multi-dimensional protein separation channelsprovides an indication of the proteins that are expressed e.g., in acell or tissue type.

In addition to identifying expression products, such as proteins or RNA,it is also possible to screen for large numbers of metabolites in cellor tissue samples. The presence, absence or level of a metabolite can betreated as a character for comparison purposes in the same way thatnucleic acids or proteins are discussed herein. Metabolites can bemonitored by any of currently available microfluidic method, includingchromatography, uni or multi dimensional gel separations, hybridizationto complementary molecules, or the like.

Immunoassays

A particular protein or other biological component can be quantified bya variety of immunoassay methods which can be practiced using the arraysof the invention. For a review of immunological and immunoassayprocedures in general, see Stites and Terr (eds.) 1991 Basic andClinical Immunology (7th ed.); Maggio (ed.) (1980) Enzyme ImmunoassayCRC Press, Boca Raton, Fla.; Tijan (1985) “Practice and Theory of EnzymeImmunoassays,” Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers B.V., Amsterdam; Harlow and Lane,supra; Chan (ed.) (1987) Immunoassay: A Practical Guide Academic Press,Orlando, Fla.; Price and Newman (eds.) (1991) Principles and Practice ofImmunoassays Stockton Press, NY; Ngo (ed.) (1988) Non isotopicImmunoassays Plenum Press, NY and Fundamental Immunology, Third Edition,W. E. Paul, ed., Raven Press, N.Y. (1993).

Immunoassays often utilize a labeling agent to specifically bind to andlabel the binding complex formed by the capture agent and the analyte.The labeling agent may itself be one of the moieties comprising theantibody/analyte complex. Thus, the labeling agent may be a labeledanalyte or a labeled anti-analyte antibody. Alternatively, the labelingagent may be a third moiety, such as another antibody, that specificallybinds to the antibody/analyte complex, or to a modified capture group(e.g., biotin) which is covalently linked to the analyte or anti-analyteantibody. Any of these components can be e.g., fixed to array members orcan be present in reagent trains which are flowed across array members.

In one embodiment, the labeling agent is an antibody that specificallybinds to a capture agent (e.g., an antibody which binds either theanalyte, an analyte-antibody complex or an antibody which binds theanalyte). Such labeling agents are well known to those of skill in theart, and most typically comprise labeled antibodies that specificallybind antibodies of the particular animal species from which the captureagent is derived (e.g., an anti-idiotypic antibody). Thus, for example,where the capture agent is a mouse derived anti-marker gene antibody,the label agent may be a goat anti-mouse IgG, i.e., an antibody specificto the constant region of the mouse antibody. Other proteins capable ofspecifically binding immunoglobulin constant regions, such asstreptococcal protein A or protein G are also used as the labelingagent. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non immunogenic reactivitywith immunoglobulin constant regions from a variety of species. See,generally Kronval, et al., (1973) J. Immunol., 111:1401-1406, andAkerstrom, et al., (1985) J. Immunol., 135:2589-2542.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about0.5 seconds to several hours. However, the incubation time will dependupon the assay format, analyte, volume of solution, concentrations, andthe like. One advantage of the present invention is that incubationtimes can ordinarily be short, because, in microfluidic systems, smallfluid volumes are ordinarily used. Usually, the assays are carried outat ambient temperature, although they can be conducted over a range oftemperatures, such as 5 C to 45 C.

(i) Non Competitive Assay Formats

Immunoassays for detecting an analyte can be, e.g., competitive ornoncompetitive. Noncompetitive immunoassays are assays in which theamount of captured analyte is directly measured. In a “sandwich” assay,for example, the capture agent (e.g., an antibody) is bound directly toan array member where it is fixed or immobilized. These immobilizedantibodies then capture analytes present in a test sample. The analyteswhich are immobilized are optionally bound by a labeling agent, such asa second antibody bearing a label. Alternatively, the second marker geneantibody may lack a label, but it may, in turn, be bound by a labeledthird antibody specific to antibodies of the species from which thesecond antibody is derived. In another format, a sandwich assay isunnecessary because either the antibody or analyte are labeled, with thecomplementary component typically being fixed to an array member.

Sandwich assays for an analyte are optionally constructed. As describedabove, the antibody or antibody ligand bound to an array memberspecifically binds to the corresponding element present in a sample. Alabeled antibody then binds to analyte-antibody complexes. Free labeledantibody is washed away, e.g., by electrophoresis, electroosmosis,electrokinesis or pressure based fluid movement and the remaining boundlabeled complex is detected (e.g., using a gamma detector where thelabel is radioactive, or an optical arrangement where the label isfluorescent or luminescent).

(ii) Competitive Assay Formats

In competitive assays, the amount of analyte present in the sample ismeasured indirectly by measuring the amount of an added (exogenous)analyte displaced (or competed away) from a capture agent by the analytepresent in the sample. In one competitive assay, a known amount ofanalyte is added to the sample and the sample is contacted with acapture agent, in this case an antibody that specifically binds theanalyte. The amount of analyte bound to the antibody is inverselyproportional to the concentration of analyte present in the sample.

In one embodiment, the capture agent is immobilized on a solidsubstrate. The amount of e.g., polypeptide bound to the capture agent isdetermined either by measuring the amount of analyte present in ananalyte-antibody complex, or alternatively by measuring the amount ofremaining uncomplexed analyte or antibody. The amount of material in asample to be assayed can also be detected by providing exogenous labeledmarker gene to the assay.

A hapten inhibition assay is another preferred competitive assay. Inthis assay, a known analyte is fixed on an array member. A known amountof antibody is added to the sample, and the sample is then contactedwith the fixed analyte. In this case, the amount of antibody bound tothe fixed analyte is proportional to the amount of analyte in thesample. Again the amount of immobilized antibody is detected byquantitating either the immobilized fraction of antibody or the fractionof the antibody that remains in solution. Detection may be direct wherethe antibody is labeled, or indirect where a labeled moiety issubsequently added which specifically binds to the antibody as describedabove.

Many other immunoassay formats are known and can be practiced by fixingone or more component of the assay to an array member in a microfluidicarray of the invention, e.g., using the coupling techniques describedsupra.

Downstream Separations

For all of the sequencing and PCR methods noted above, as well as formany other methods noted herein, products can be electrophoresed, e.g.,following release from an array, e.g., to facilitate separation anddetection of the products. microfluidic systems which combine fluidhandling and electrophoresis are described, e.g., in U.S. Ser. No.09/093,832 (now U.S. Pat. No. 6,306,590) “MICROFLUDIIC MATRIXLOCALIZATION APPARATUS AND METHODS” Burd Mehta and Kopf-Sill filed Jun.8, 1998. In brief, this application, which describes, e.g., anaphasicand especially multiphasic microfluidic systems, a channel comprising aliquid phase intersects a channel comprising a sieving matrix. Appliedto the present invention, products are optionally washed from arrays,where they are flowed into contact with a sieving matrix. The productcomponents typically “stack” at the fluid-sieving matrix interface, andare then electrophoresed through the sieving matrix. The products canthen be detected during or after electrophoresis, e.g., by placing adetection element within or proximal to the sieving matrix. The productscan also be purified in the sieving matrix and electrokinetically (or bypressure mechanisms) moved into contact with subsequent reactants oradditional array members for further processing or for use as reactantsin subsequent reactions (e.g., as templates in sequencing reactions, astargets for amplification, as probes to detect amplified products orother targets, etc.).

In uniphasic separatory systems, array components are optionallydispersed within a sieving matrix. Reactions such as sequencing, PCR,LCR, or the like can be conducted on or proximal to the array members,with products being released into the sieving matrix for separation.

In addition to the multiphasic and uniphasic microfluidic systems of the'832 application, a variety of microfluidic electrophoretic applicationsare described, e.g., in “Closed Loop Biochemical Analyzers” (WO98/45481), as well as other references available in the art.

Type Switchable Arrays

In one aspect, the arrays are particle type switchable. In thisembodiment, arrays are modified by flowing reagents across all, or aportion of the particle sets of the arrays. The reagents chemicallyinteract with (e.g., covalently modify, hybridize to, or the like)particle sets of the array, thereby altering one or more sets of thearray. Thus, particle sets can be fixed in place (or flowable) andswitchable from one type of particle set to another. For example, in oneaspect, the ordered array of a plurality of sets of particles isproduced by flowing a substantially homogeneous or heterogeneous set ofparticles into a particle modification region and flowing a plurality ofparticle modification reagents across the substantially homogeneous orheterogeneous set of particles. The reagents react with thesubstantially homogeneous or heterogeneous set of particles to create aplurality of sets of different particles. For example, the homogeneousor heterogeneous sets of particles optionally include a plurality ofparticles which have one or more molecular tags (e.g., streptavidin,avidin, biotin, an antibody, an antibody ligand, a nucleic acid, anucleic acid binding molecule, etc.). The plurality of particlemodification reagents can include one or more anti-tag ligand. Theplurality of particle modification reagents can be flowed sequentiallyacross the substantially homogeneous set of particles, thereby bindingthe anti-tag ligand to the tag and producing sets of differentparticles, each set having a different bound particle modificationreagent such as a nucleic acid. Tags and tag ligands can be attached toparticles or particle modification reagents directly or through alinker, through covalent or non-covalent interactions.

The ability to switch the type of a particle member of an array providesan elegant method for making arrays within microfluidic systems. Inparticular, arrays are made in situ by exposure to reagents, avoidingthe necessity of moving and tracking different particle sets todifferent array locations.

Moreover, particle type switchability provides for the creation ofmicrofluidic logic circuits. In particular, the presence or absence of asignal from an array location is equivalent to a bit of information in atypical computer system and it is possible to reprogram the array simplyby flowing appropriate reagents to appropriate array positions. Itshould be appreciated that, in at least one sense, these switchablearrays are superior to existing silicon-based computer design. Inparticular, rather than being limited to a simple digital “on/off”binary programming language, it is possible to obtain analogueinformation from the arrays. This is because degrees of signal intensityfrom array locations can be discerned. Thus, a very rich non-binaryprogramming language can be used in programming and interpretingswitchable microfluidic arrays. Of course, switchable arrays canincorporate both ordinary binary silicon-based switches, as well asmicrofluidic switches, providing for design heuristics that incorporateboth binary and non-binary programming.

In addition to the creation of logic circuits and array construction,type-switchable arrays can be used as chemical synthesis andpurification machines. In particular, array members can have chemicalcomponents synthesized in a solid phase fashion on the array members,and subsequently used to purify complementary molecules in a mannersimilar to affinity chromatography. Alternatively, following synthesis,chemical moieties can be cleaved from the array for subsequent use inother microfluidic assays (or even for purification and use outside ofthe microfluidic system). This ability to act as a biochemical reactoris a preferred aspect of the present invention.

Reagent Caging and Triggering

The present invention provides for reagent triggering upon contact withan array member. For example, certain reagents are activated by heat orcold, or are activated by changes in pH, light levels, or the like.These reagents can be maintained in an inactive state and activated whenthey are brought into contact with an array member. Similarly, arraymembers can be maintained in an inactive state and activated by exposureto a temperature change, a change in pH, light, or the like.

Similarly, reagents can be trapped or “caged” by being complexed to aparticle and released or “uncaged” from the particle by exposure to anactivating reagent or reaction condition. Using these approaches,reagents can be delivered in trapped or inactive packets to a reactionsite where they are released and/or activated.

For example, a sequencing reagent such as a nucleic acid template can befixed to a particle, e.g., by synthesizing or fixing a single-strandedoligonucleotide on the surface of the particle and hybridizing acomplimentary nucleic acid to the fixed single stranded nucleic acid.The complimentary nucleic acid can be released by exposure to, e.g.,heat, or exposure to a base (e.g., dilute NaOH), or exposure to adenaturant (e.g., guanidine HCL). For example, the particle isoptionally flowed to a point where a sequencing reaction is to beperformed, and the complimentary strand then released by heat orexposure to base or a denaturant. Alternatively, a sequencing reaction,e.g., by PCR exonuclease digestion, is performed on one or more DNAtemplate attached to a particle, which particle is fixed in the channel,e.g., by a particle retention element, by another set of particles, orthe like. The DNA is then optionally released after sequencing forseparation or further manipulation. Similarly, proteins can beassociated (e.g., by bonding to fixed ligands or antibodies) andreleased (e.g., by exposure to heat or a base or a denaturant) from theparticle. Other molecules such as polymers or large organic moleculescan similarly be caged and released from particle members.

Similarly, reagents such as enzymes (e.g., thermostable enzymes),sequencing reagents, or other molecules can be held in an inactive form(e.g., due to heat or presence of blocking groups on the molecules,e.g., those set forth in Greene, et al. (1991) Protective Groups InOrganic Chemistry, 2nd Ed., John Wiley & Sons, New York, N.Y.) andflowed into contact with array members. The reagents or other moleculescan then be activated, e.g., by exposure to light, changes intemperature, exposure to an acid, base, or a denaturant, or the like.Thus, in one embodiment, reagents are held in a reagent train in aninactive state and are activated only upon exposure to an appropriateactivation agent (heat, light, base, acid, denaturant, electric ormagnetic field, etc.).

Modulation of Hydrodynamic Resistance with Particle Sets; IterativeFluid Manipulations

The hydrodynamic resistance in a channel can be increased by packing thechannel with particles. In addition, electroosmotic flow can be alteredsignificantly by packing the capillary with particles having differentsurface charges and thus different zeta potential. In one aspect of thepresent invention, particle manipulation in channels can be used todynamically control the resistance to pressure or electrokinetic flow toenhance the flexibility of microfluidic operations.

In particular, microbeads or other particles are transported intomicrochannels by any of the flow methods described herein. The particlesare captured (by a physical barrier, electric field, magnetic field,porous matrix, sintered glass frit, a fixed set of particles, etc., asalso described supra). By localizing particles to a selected location,it is possible to alter the local zeta potential, surface charge, etc.This, in turn alters flow characteristics in the region, providing foralterations in microfluidic operations, e.g., changes in the rate, oreven direction, of fluid flow. Thus, in addition to interacting withcomponents of various reactions, particle sets can also be used tomodulate fluid flow in a selected region.

Similarly, a powerful application of this dynamic microfluidic controlis to create a “smart” microfluidic system. Applications which useparticles in assays are described herein. The combination of flowmodulation by microbead localization and e.g., chemical assays on thebeads provides for a great deal of flexibility in designing andcontrolling assays. In addition, with appropriate software feedbackcontrol, subsequent assay steps are selectable based upon the results ofinitial assays. This ability to reconfigure flow conditions and assaycomponents by manipulating particles in response to assay results is avery potent new way of performing iterative reactions.

For example, the integrated assay and flow control features provide veryhigh throughput methods of assessing biochemical components andperforming biochemical manipulations. A wide variety of reagents andproducts are suitably assessed, including libraries of chemical orbiological compounds or components, nucleic acid templates, PCR reactionproducts, and the like. In the integrated systems it is possible to usethe results of a first reaction or set of reactions to selectappropriate flow conditions, reagents, reactants, products, or the like,for additional analysis. For example, the results of a first sequencingreaction can be used to select primers, templates or the like foradditional sequencing, or to select related families of compounds forscreening in high-throughput assay methods. These primers or templates(e.g., as components of an array) are then accessed by the system andthe process continued.

In one aspect, the invention provides integrated methods of analyzingand manipulating sample materials for fluidic analysis on arrays. In themethods, an integrated microfluidic system including a microfluidicdevice comprising an array is provided. The device has at least a firstreaction channel and at least a first reagent introduction channel,typically etched, machined, printed, or otherwise manufactured in or ona substrate. Optionally, the device can have a second reaction channeland/or reagent introduction channel, a third reaction channel and/orreagent introduction channel or the like, up to and including hundredsor even thousands of reaction and/or reagent introduction channels. Thereaction channel and reagent introduction channels are in fluidcommunication, i.e., fluid can flow between the channels under selectedconditions. The device has a material transport system for controllablytransporting a material through and among the reagent introductionchannel and reaction channel and for positioning array components. Forexample, the material transport system can include electrokinetic,electroosmotic, electrophoretic or other fluid manipulation aspects(micro-pumps and microvalves, fluid switches, fluid gates, etc.) whichpermit controlled movement and mixing of fluids and movement of arraymembers. The device also has a fluidic interface in fluid communicationwith the reagent introduction channel. Such fluidic interfacesoptionally include capillaries, channels, pins, pipettors,electropipettors, or the like, for moving fluids, and optionally furtherinclude microscopic, spectroscopic, fluid separatory or other aspects.The fluidic interface samples a plurality of reagents or mixtures ofreagents from a plurality of sources of reagents or mixtures of reagentsand introduces the reagents or mixtures of reagents into the reagentintroduction channel. Essentially any number of reagents or reagentmixtures and or array members can be introduced by the fluidicinterface, depending on the desired application. Because microfluidicmanipulations are performed in a partially or fully sealed environment,contamination and fluidic evaporation in the systems are minimized.

In the methods, a first reagent from the plurality of sources of reagentor mixtures of reagents is selected. A first sample material and thefirst reagent or mixture of reagents is introduced into the firstreaction channel, whereupon the first sample material and the firstreagent or mixture of reagents react. Typically, one of the first samplematerial or first reagent or reagent mixture are bound to amicroparticle in an array. This reaction can take a variety of differentforms depending on the nature of the reagents. For example, where thereagents bind to one another, such as where the reagents are an antibodyor cell receptor and a ligand, or an amino acid and a binding ligand,the reaction results in a bound component such as a bound ligand (e.g.,bound to an array member). Where the reagents are sequencing reagents, aprimer extension product results from the reaction. Where the reagentsinclude enzymes and enzyme substrates, a modified form of the substratetypically results. Where two reacting chemical reagents are mixed, athird product chemical typically results.

In the methods, a reaction product of the first sample material and thefirst reagent or mixture of reagents is analyzed in the context of anarray. This analysis can take any of a variety of forms, depending onthe application. For example, where the product is a primer extensionproduct, the analysis can take the form of separating reactants by size,or by location on the array and detecting the sized reactants andtranslating the resulting information to give the sequence of a templatenucleic acid. Similarly, because microscale fluidic devices of theinvention are optionally suitable for heating and cooling a reaction, aPCR reaction utilizing PCR reagents (thermostable polymerase,nucleotides, templates, primers, buffers and the like) can be performedand the PCR reagents detected. Where the reaction results in theformation of a new product, such as an enzyme-substrate product, achemical species, or an immunological component such as a bound ligand,the product is typically detected by any of a variety of detectiontechniques, including fluorescence, autoradiography, microscopy,spectroscopy, or the like.

Based upon the reaction product, a second reagent or mixture of reagentsis selected and a second sample material is assessed, optionallyfollowing manipulation of particle sets to modify flow conditions. Forexample, where the product is a DNA sequence, a sequencing primer and/ortemplate for extension of available sequence information is selected.Where the product is a new product such as those above, an appropriatesecond component such as an enzyme, ligand, antibody, receptor molecule,chemical, or the like, is selected to further test the binding orreactive characteristics of an analyzed material. The second reagent ormixture of reagents is introduced into the first reaction channel, oroptionally into a second (or third or fourth . . . or nth) reactionchannel in the microfluidic device. The second sample material and thesecond reagent or mixture of reagents react, forming a new product,which is analyzed as above. The results of the analysis can serve as thebasis for the selection and analysis of additional reactants andadditional flow conditions for similar subsequent analysis. The secondsample material, reagents, or mixtures of reagents can comprise the sameor different materials. For example, a single type of DNA template isoptionally sequenced in several serial reactions. Alternatively,completing a first sequencing reaction, as outlined above, serves as thebasis for selecting additional templates (e.g., overlapping clones, PCRamplicons, or the like).

Accordingly, in a preferred aspect, the invention provides methods ofsequencing a nucleic acid by an iterative process on an array. Forexample, in one typical method, the biochemical components of asequencing reaction (e.g., a target nucleic acid, a first andoptionally, second sequencing primer, a polymerase (optionally includingthermostable polymerases for use in PCR), dNTPs, and ddNTPs) are mixedin a microfluidic device in contact with one or more array member underconditions permitting target dependent polymerization of the dNTPs.Polymerization products are optionally separated in the microfluidicdevice to provide a sequence of the target nucleic acid, or as in thecase of pyrophosphate methods described above, can be read directly fromthe array, depending on the position of components in the array(similarly, sequencing by hybridization methods do not requireseparation of products, with results being determined by position on anarray). Typically, sequencing information acquired by this method isused to select additional sequencing primers and/or templates or probes,and the process is reiterated, optionally following movement of arraycomponents to modulate flow conditions.

In one integrated sequencing system, methods of sequencing a targetnucleic acid are provided in which an integrated microfluidic systemcomprising a microfluidic device having an array is utilized in thesequencing method. The integrated microfluidic device has at least afirst sequencing reaction channel and at least a first sequencingreagent introduction channel, the sequencing reaction channel andsequencing reagent introduction channel being in fluid communication andat least one of the channels comprising one or more array component. Theintegrated microfluidic system also has a material transport system forcontrollably transporting sequencing reagents through the sequencingreagent introduction channel and sequencing reaction channel and afluidic interface in fluid communication with the sequencing reagentintroduction channel for sampling a plurality of sequencing reagents, ormixtures of sequencing reagents, from a plurality of sources ofsequencing reagents or mixtures of sequencing reagents and introducingthe sequencing reagents or mixtures of sequencing reagents into thesequence reagent introduction channel. For example, the system set forthin U.S. Pat. No. 5,779,868 can be used. As discussed above, theinterface optionally includes capillaries, pins, pipettors and the like.In the method, a first sequencing primer sequence complementary to afirst subsequence of a first target nucleic acid sequence is introducedinto the sequence reagent introduction channel. The first primer ishybridized to the first subsequence and the first primer is extendedwith a polymerase enzyme along the length of the target nucleic acidsequence to form a first extension product that is complementary to thefirst subsequence and a second subsequence of the target nucleic acid.Again, any of these components are optionally coupled to one or morearray member. The sequence of the first extension product is determinedand, based upon the sequence of the first extension product, a secondprimer sequence complementary to a second subsequence of the targetnucleic acid sequence is selected, hybridized and extended as above.

In the sequence methods herein, it is sometimes advantageous to selectsequencing primers from a large set of sequencing primers, rather thansynthesizing primers to match a particular target nucleic acid. Forexample, 5 or 6-mer primers can be made to hybridize specifically to atarget, e.g., where the primers are modular and hybridize to a singleregion of a nucleic acid. All possible 5 or 6 mers can be synthesizedfor selection in the methods herein, or any subset of 5 or 6 mers canalso be selected. In some embodiments, the primers are transferred tothe microfluidic apparatus, e.g., by a capillary, an electropipettor, orusing sipping technology, from a microtiter plate or from and array ofoligos. The primers are used to hybridize to bound targets fixed toarray members (see also, above, under discussions of modular primerstrategies). In other embodiments, the primers are located on a regionof a microfluidic device, chip or other substrate.

In another, similar aspect, the devices, systems arrays and methods ofthe invention are useful in performing fluidic operations that require alarge number of successive fluid manipulations, i.e., in performing anumber of preparative and analytical reactions or operations on a givensample. By “successive fluid manipulations” is generally meant a fluidicoperation that involves the successive treatment of a given fluid samplevolume, i.e., combination/reaction with reactants, incubation,purification/separation, analysis of products, and the like. Wheresuccessive fluid manipulations are performed at the bench scale, e.g.,the performance of numerous, different manipulations on a particularsample such as combination with reagents, incubation, separation anddetection, such manipulations can also become cumbersome as the numberof steps increases, as with each step, the possibility of introducing anerror into the operation or experiment increases. This complexity, andthe consequent increased possibility of errors increases substantiallyas the number of samples to be passed through the operation increases.Thus, the devices or systems of the present invention are alsoparticularly useful in performing fluidic operations which requiresuccessive fluid manipulations of a given sample or fluid of interest,e.g., more than 2 steps or different manipulations, typically greaterthan 5 steps or different manipulations, preferably greater than 10steps or different fluid manipulations. The systems are also useful andreadily capable of performing fluidic operations that include greaterthan 20, 50, 100, 1000 steps or different fluid manipulations on a givenfluid volume.

In a related, but alternate aspect, the devices, arrays, systems andmethods of the invention are useful in performing fluidic operationsthat require a large number of parallel fluid manipulations, i.e., toscreen biological samples, screen test compounds for drug discovery,e.g., as set forth in WO/98/00705 and WO 98/00231 and incorporatedherein by reference. To carry out these operations, a substrate willtypically employ parallel channels and/or channel networks,interconnected by one or more common channels, with at least oneparticle array dispersed within the device. Fluids required for thesubject reaction, e.g., samples or reagents, are directed along one ormore of the common channels, and are delivered to each of the parallelchannels.

As used herein, “parallel fluid manipulations” means the substantiallyconcurrent movement and/or direction, incubation/reaction, separation ordetection of discrete fluid volumes to a plurality of parallel channelsand/or channel networks, or chambers of a microfluidic device, i.e.,greater than about 10 distinct parallel channels or chambers, typicallygreater than 20 distinct channels or chambers, preferably greater thanabout 50 distinct channels or chambers, and often greater than about 100distinct channels or chambers. As used herein, the term “parallel”refers to the ability to concomitantly or substantially concurrentlyprocess two or more separate fluid volumes, and does not necessarilydenote a specific channel or chamber structure or layout.

Ultra high-throughput analysis systems are provided, for example forperforming nucleic acids-based diagnostic and sequencing applications,e.g., in a reference laboratory setting. The system typically hasseveral components: a specimen and reagents handling system; an“operating system” for processing integrated microchip experimentationsteps; application-specific analysis devices; a signal detection system,and multiple software components that allow the user to interact withthe system, and run processing steps, interpret data, and reportresults.

Integrated Systems for Assay Normalization

One similar application of the integrated systems and arrays of theinvention is the titration of assay components into the dynamic range ofan assay. For example, an assay can first be performed where one or morecomponents of the assay are not within the range necessary for adequateperformance of the assay, e.g., if the assay is performed using aconcentration which is too high or too low for some components, theassay may not provide quantitative results. This need to titrate assaycomponents into the dynamic range of an assay typically occurs where oneor more component of the assay is present at an unknown activity orconcentration. Ordinarily, the assay must be run at severalconcentrations of components, i.e., the assay is run a first time,components are diluted, the assay is run a second time, etc. until theassay can be performed within the dynamic range of the assay. It will beappreciated that this iterative approach can involve several unknownconcentrations simultaneously, requiring considerable trial and error.

In the integrated array systems of the invention, an assay can beperformed at as many concentrations of components as necessary totitrate the assay components into the dynamic range of the assay, withthe results of each assay being used to optimize additional assaypoints. Similarly, titration curves, which are often the result ofmultiple assay runs with different component concentrations aredetermined by performing repeated assays with different concentrationsof components. Different concentrations of assay components in separateassays can be monitored serially or in parallel. In brief, one simplyruns the assay at one or more array location, detecting the results. Ifassays are simultaneously (or even separately) run at additional arraylocations with known components, it is possible to use these knowncomponents as normalization elements for the array and the assay. Thus,arrays can include positive or negative control elements (reagents,templates, etc.), calibration components (e.g., an array can include alabeled array member and an unlabeled array member for calibrationpurposes, or even include members comprising gradations of labelintensity).

For example, in one aspect, the invention comprises any of “blank,”“dummy,” “calibration,” “control,” “positive control,” “negativecontrol,” “sample,” “test” or “tracking” particles, e.g., interspersedwith each other or with other elements of an array. Blank particles areparticles which do not comprise a label. Dummy particles are eitherblank, or comprise a known signal component. Calibration particlescomprise a selected quantity of labeled component, or of a component tobe labeled. For example, calibration particles (or any of the otherparticles herein) can comprise one or more quantum dot (Warren and Nie(1998) Science, 281: 2016-2018). Control particles comprise one or moreselected known element. Positive control particles comprise a knowncomponent which, if the assay or other use for the array member isworking properly, will result in accumulation or display of a detectablesignal. Conversely, a negative control particle is a particle that, ifthe assay or other use for the array member is working properly will notresult in a significant accumulation, release or display of a detectablesignal. A “sample” particle comprises an assay or reaction element ofinterest. A “test” particle is a particle that comprises an unknownelement to be tested, or which interacts with such an element in anassay or reaction, depending on the specified context of the invention.A “tracking” particle is a particle which displays a detectable signal,or which displays a known absence of signal within a labeled array, andwhich is used to track the position of array elements.

It will be appreciated that many useful particle types will meet morethan one of the above criteria. For example a negative control particlecan also be a blank, a tracking element, or the like. Furthermore, theparticles can be placed in any selected relative positionalconformation. For example, blank particles can be interspersed withdifferent sample particles to maintain separation and preventcontamination between the different sample sets. Calibration particlescan be interspersed with sample particles in any manner to providesignals for calibration of assays, reactions, etc. Many such variationswill be apparent upon review.

The ability to titrate and optimize assays is useful for diagnosticassays, for determining concentrations or activities of selectedcomponents in a system (proteins, enzymes, nucleic acids, smallmolecules, etc.). Furthermore, the present integrated systems providefor rational selection of assay conditions as data is acquired. Forexample, in one embodiment, a diagnostic assay needs to be performedusing several components which are present at initially unknownconcentrations or activities. A first series of concentration oractivity assays is performed on the array to determine the activity orconcentration of particular components, e.g., enzyme, protein,inhibitor, co-factor, nucleic acid, or the like. After these assays areperformed and the concentrations or activities of some or all of thecomponents for the diagnostic assay are determined, the integratedsystem selects appropriate amounts of the assay components, performs anynecessary dilutions, combines the assay components and performs thediagnostic assay. Similarly, further data points can be collected byadjusting the concentrations or amounts of diagnostic assay componentsand re-running the assay. All of the fluid manipulations can beperformed rapidly and the integrated system is able to assess andcompile the results of individual data points or individual assays toselect which additional assays need to be performed for assayverification.

In its most basic form, assay optimization involves the identificationof factors affecting a reaction result, followed by the systematicvariation of each of these variables until optimal reaction conditionsare identified. This is generally termed an “OFAT” method for “onefactor at a time.” Thus, assuming a simple two reagent reaction, onewould first identify the factors affecting the outcome, e.g.,concentration of reagent A, concentration of reagent B and, e.g.,temperature. One would then run the assay where one factor was variedwhile the others remained constant. For example, one would perform thesame reaction at numerous different concentrations of reagent A, whilemaintaining the concentration of reagent B and the temperature. Next,reagent B would be varied while reagent A and temperature remainedconstant, and finally, the temperature would be varied.

Even in this simplest form, the number and complexity of necessaryreactions is apparent. When one considers that most reactions will havefar more than three variables, and that these variable will not beindependent of each other, the possibility of manually performing theseassays, or even performing them in currently available automated formatsbecomes a daunting prospect. For example, while robotic systems usingmicrowell plates can perform large numbers of manipulations to optimizeassay parameters, such systems are very expensive. Further, as thesesystems are typically limited to the bench scale volumes describedabove, they require large volumes of reagents and large amounts of spacein which to operate.

In contrast, the devices, systems and methods of the present inventionpermit the optimization of large numbers of different assays, byproviding an extremely low volume, automatable and sealed format inwhich such optimization can occur rapidly and automatically. Forexample, the devices can run a first fluidic operation by combining apreselected volume of a first reactant with a preselected volume of asecond reactant, at a desired or preselected temperature for a desiredor preselected amount of time. The device then repeats the assay, butvaries at least one of the volume of the first or second reactants, thetemperature, or the amount of time allowed for the reaction. This isrepeated until a desired number of varied reactions are performed, i.e.,generating sufficient data to permit an estimation of optimal assayconditions which will produce an optimal result of the reaction, withina desired range of statistical significance. “optimal assay conditions”include those conditions that are required to achieve the desired resultof the reaction. Such desired results can include maximization ofreaction yields, but also includes assay conditions which are optimizedfor sensitivity to one variable, e.g., inhibitor concentration, and thelike.

Drug Screening Assays

In addition to sequencing, the integrated microfluidic systems andarrays of the invention are broadly useful in a variety of screeningassays where the results of mixing one or more components are to bedetermined, and particularly, where the results determined are used toselect additional reagents to be screened.

As described more fully below, the integrated microfluidic system of theinvention can include a very wide variety of storage elements forstoring reagents to be assessed. These include well plates, matrices,membranes and the like. The reagents are stored in liquids (e.g., in awell on a microtiter plate), or in lyophilized form (e.g., dried on amembrane), and can be transported to an array component of themicrofluidic device using conventional robotics, or using anelectropipettor as described below.

Because of the breadth of the available sample storage formats for usewith the present invention, virtually any set of reagents can be sampledand assayed in an integrated system of the present invention. Forexample, enzymes and substrates, receptors and ligands, antibodies andligands, proteins and inhibitors, cells and growth factors orinhibitors, viruses and virus binding components (antibodies, proteins,chemicals, etc.) immunochemicals and immunoglobulins, nucleic acids andnucleic acid binding chemicals, proteins, or the like, reactantchemicals (acids, bases, organic molecules, hydrocarbons, silicates,etc.) can all be assayed using the integrated systems of the invention.For example, where a molecule which binds a protein is desired,potential binding moieties (chemicals, peptides, nucleic acids, lipids,etc.) are sequentially mixed with the protein in a reaction channel, andbinding is measured (e.g., by change in electrophoretic mobility,quenching of fluorescent protein residues, or the like). Thousands ofcompounds are easily screened using this method, in a short period oftime (e.g., less than an hour).

An advantage of the integrated nature of the present system is that itprovides for rational selection of structurally or functionallyhomologous compounds or components as the assay progresses. For example,where one compound is found to have binding activity in an array basedassay, the selection of a second compound to be tested can be performedbased upon structural similarity to the first active compound.Similarly, where a compound is shown to have activity in a cell (e.g.,up-regulation of a gene of interest) a second compound affecting thesame cellular pathway (e.g., calcium or inositol phosphate secondmessenger systems, etc.) can be selected from the group of availablecompounds for testing. In this way, it is possible to focus screeningassays from purely random at the outset to increasingly focused onlikely candidate compounds as the assays progress.

Integrated Microfluidic Array Systems

Device Integration

Although the devices and systems specifically illustrated herein aregenerally described in terms of the performance of a few or oneparticular operation, it will be readily appreciated from thisdisclosure that the flexibility of these systems permits easyintegration of additional operations into these devices. For example,the devices and systems described will optionally include structures,reagents and systems for performing virtually any number of operationsboth upstream and downstream from the operations specifically describedherein. Such upstream operations include sample handling and preparationoperations, e.g., cell separation, extraction, purification,amplification, cellular activation, labeling reactions, dilution,aliquoting, and the like. Similarly, downstream operations may includesimilar operations, including, e.g., separation of sample components,labeling of components, assays and detection operations, electrokineticor pressure-based injection of components into contact with particlesets, or materials released from particle sets, or the like.

Assay and detection operations include, without limitation, probeinterrogation assays, e.g., nucleic acid hybridization assays utilizingindividual probes, free or tethered within the channels or chambers ofthe device and/or probe arrays having large numbers of different,discretely positioned probes, receptor/ligand assays, immunoassays, andthe like. Any of these elements can be fixed to array members, or fixed,e.g., to channel walls, or the like.

Loading of Array Components and Reagents

Array members and reagents can be loaded into microfluidic structures,e.g., by placing the reagent or array member in a well fluidly coupledto a microfluidic channel network. The reagent or array member is thenflowed through the microchannel network as described supra, e.g., bypressure (positive or negative) or by electrokinesis, or by moving amagnetic field relative to the array member (i.e., where the arraymember is magnetic).

Alternatively, array or particle members can be stored external to themicrofluidic system in a system of wells, plates, or even as driedcomponents stored on a surface. Thus, the integrated systems of theinvention optionally include such external storage elements. In oneaspect, the present invention includes a microwell plate (e.g., a 96,384 or more well plate) having array members stored within wells of theplate.

To introduce array members or reagents into the microfluidic system,either pressure-based, electrokinetic or centrifugal approaches can beused. For example, electropipettors (which can include one or multiple“sipper” channels) can be used to access wells, plates or to re-hydratesoluble or suspendable dried components from dry storage media. Avariety of access systems for coupling reagent storage and microfluidicsystems are described in Knapp et al. “Closed Loop BiochemicalAnalyzers” WO 98/45481. As applied to the present invention, thesecoupling devices and strategies are also used for flowing particle setsinto microfluidic systems.

Instrumentation

In the present invention, the materials in the arrays are optionallymonitored and/or detected so that velocity can be determined. Fromvelocity measurements, decisions are then made regarding subsequentfluidic operations.

The systems described herein generally include microfluidic devices, asdescribed above, in conjunction with additional instrumentation forcontrolling fluid transport, flow rate and direction within the devices,detection instrumentation for detecting or sensing results of theoperations performed by the system, processors, e.g., computers, forinstructing the controlling instrumentation in accordance withpreprogrammed instructions, receiving data from the detectioninstrumentation, and for analyzing, storing and interpreting the data,and providing the data and interpretations in a readily accessiblereporting format.

Controllers

A variety of controlling instrumentation is optionally utilized inconjunction with the microfluidic devices described above, forcontrolling the transport and direction of fluids and/or materialswithin the devices of the present invention, e.g., by pressure-based orelectrokinetic control.

For example, in many cases, fluid transport and direction are controlledin whole or in part, using pressure based flow systems that incorporateexternal or internal pressure sources to drive fluid flow. Internalsources include microfabricated pumps, e.g., diaphragm pumps, thermalpumps, lamb wave pumps and the like that have been described in the art.See, e.g., U.S. Pat. Nos. 5,271,724, 5,277,556, and 5,375,979 andPublished PCT Application Nos. WO 94/05414 and WO 97/02357. In suchsystems, fluid direction is often accomplished through the incorporationof microfabricated valves, which restrict fluid flow in a controllablemanner. See, e.g., U.S. Pat. No. 5,171,132.

As noted above, the systems described herein can also utilizeelectrokinetic material direction and transport systems. As such, thecontroller systems for use in conjunction with the microfluidic devicestypically include an electrical power supply and circuitry forconcurrently delivering appropriate voltages to a plurality ofelectrodes that are placed in electrical contact with the fluidscontained within the microfluidic devices. Examples of particularlypreferred electrical controllers include those described in, e.g.,published PCT application WO 98/00707 and in U.S. Pat. No. 5,800,690. Inbrief, the controller uses electric current control in the microfluidicsystem. The electrical current flow at a given electrode is directlyrelated to the ionic flow along the channel(s) connecting the reservoirin which the electrode is placed. This is in contrast to the requirementof determining voltages at various nodes along the channel in a voltagecontrol system. Thus the voltages at the electrodes of the microfluidicsystem are set responsive to the electric currents flowing through thevarious electrodes of the system. This current control is lesssusceptible to dimensional variations in the process of creating themicrofluidic system in the device itself. Current control permits fareasier operations for pumping, valving, dispensing, mixing andconcentrating subject materials and buffer fluids in a complexmicrofluidic system. Current control is also preferred for moderatingtemperature effects (e.g., joule heating) within the channels.

The present invention also provides novel methods of controlling fluidflow, particle flow, or the like in microfluidic channels. By flowingreagents, samples, and/or particle arrays through a microfluidic networkusing a system of split wells, contamination by components of previousreactions is avoided. In an assay involving many components, thecomponents typically vary in charge. Therefore when transportingmaterials using electrokinetic techniques, components may move indifferent directions depending on their charge, especially in buffers inwhich the electroosmotic mobility is low. Therefore, a sampleintroduction step may not properly introduce all components to thereaction area and a washing step may not clear away all reactioncomponents to the waste reservoir(s) before the next reaction or phase.Therefore, some components may carry over from a first reaction to asecond reaction in a sequential system, e.g., a high throughput system.

For example, in a pyrosequencing reaction in which the DNA template isimmobilized on beads, all sequencing reagents are optionally present ina reaction mixture along with reagents used to transform any generatedpyrophosphate into chemiluminescence. The DNA sequencing reagents, e.g.,dNTPs, and the pyrophosphate are negatively charged, but some enzymescan be positively charged. The present system clears away bothpositively and negatively charged components before the next baseextension reaction begins, thus avoiding contamination between samples.

To provide more complete washing of oppositely charged components, e.g.,from a microfluidic system, a pair of wells is provided on each side ofa reaction region. Reactions of interest, e.g., pyrosequencing, occur,e.g., in a network of microscale channels or capillaries, in thereaction area. One well of each pair is used as a waste well and theother is used as a reagent source or reservoir, e.g., for reagents of acertain charge. For example, see FIG. 18, Panel A. Well 1810 comprises asource of positive and neutral reagents and well 1830 comprises a sourcefor negative reagents (or those reagents whose total electrokineticmobility is negative). Wells 1820 and 1840 comprise waste wells.Reagents are optionally loaded into the microfluidic system, e.g.,reaction area 1850, by applying a positive voltage to well 1810 relativeto well 1830. Substantially zero current is applied at wells 1820 and1840. All reagents are loaded simultaneously using this method. Positiveand neutral reagents flow from well 1810 to the reaction area 1850 andnegative reagents flow from well 1830 to reaction area 1850. Typically,the reagents are loaded quickly to avoid generation of reaction productsduring loading. Alternatively, the positive and/or neutral reagents areloaded separately from the negative reagents. Positive and neutralreagents only are loaded by applying a positive voltage at well 1810relative to well 1840 and substantially zero current at wells 1830 and1820. The negative reagents alone are loaded by applying a positivevoltage at well 1820 relative to well 1830 and substantially zerocurrent at wells 1840 and 1810. Reagents are removed from reaction area1850 in a similar manner. A positive voltage is applied to well 1820relative to well 1840 and substantially zero voltage at wells 1810 and1830. This step does not use either of the reagent wells, 1810 and 1830,thereby preventing contamination as reagents are removed or rinsed fromthe device.

Alternative channel configurations are provided in FIG. 18, panels B andC. Reaction areas 1850 b and 1850 c illustrate possible channelconfigurations. Other configurations as described herein are alsooptionally used. Wells 1810 b and 1810 c are optionally used forpositive and/or neutral reagents and wells 1830 b and 1830 c optionallycomprise negative reagents. Introduction of reagents optionallycomprises applying a positive voltage at well 1810 b or 1810 c relativeto 1830 b or 1830 c. Alternatively, a positive voltage is applied atwell 1810 b or 1810 c relative to one or more of wells 1860 b, 1860 c,1820 b, 1820 c, 1840 b, and 1840 c. Negative reagents are optionallyintroduced by applying a positive voltage at any of wells 1860 b, 1860c, 1820 b, 1820 c, 1840 b, and 1840 c relative to well 1830 b or 1830 c.Removal of reagents is achieved, e.g., by applying a positive voltage at1860 b or 1860 c relative to 1840 b or 1840 c. Other possible methods offlowing reagents through the devices of FIG. 18 will be readily apparentupon further review.

Typically, the controller systems are appropriately configured toreceive a microfluidic device as described herein. In particular, thecontroller and/or detector (as described in greater detail, below),includes a stage upon which the device of the invention is mounted tofacilitate appropriate interfacing between the controller and/ordetector and the device. Typically, the stage includes an appropriatemounting/alignment structural element, such as a nesting well, alignmentpins and/or holes, asymmetric edge structures (to facilitate properdevice alignment), and the like. Many such configurations are describedin the references cited herein.

The controlling instrumentation discussed above is also used to providefor electrokinetic injection or withdrawal of material downstream of theregion of interest to control an upstream flow rate. The sameinstrumentation and techniques described above are also utilized toinject a fluid into a downstream port to function as a flow controlelement.

Detector

The devices herein optionally include signal detectors, e.g., whichdetect fluorescence, phosphorescence, radioactivity, pH, charge,absorbance, luminescence, temperature, magnetism or the like. Thedetectors optionally monitor a plurality of signals from the pluralityof particle sets, either simultaneously or sequentially. For example,the detector can monitor a plurality of optical signals which correspondin position to sets of particles within the array. Example detectorsinclude of photo multiplier tubes, a CCD array, a scanning detector orgalvo-scann or the like. Particles from the array which emit adetectable signal can be flowed past the detector, or, alternatively,the detector can move relative to the array to determine particleposition (or, preferably, the detector can simultaneously monitor anumber of spatial positions corresponding to array members, e.g., as ina CCD array). The detector can include or be operably linked to acomputer, e.g., which has software for converting detector signalinformation into nucleic acid sequence information, converting detectorsignal information into reaction kinetic information, converting signalinformation into antibody binding data, converting signal informationinto cell receptor binding data converting signal information intohybridization data, or the like.

Signals from arrays are optionally calibrated, e.g., by calibrating themicrofluidic system by monitoring a signal from a known member of thearray, e.g., a calibration or marker particle, or from a known particleset external to the array. Similarly the relative positions of particlesets and signals from the array is monitored, e.g., by determining theposition of one or more members of the array by monitoring a signal froma known member of the array, thereby determining the position of theknown member of the array.

In the microfluidic systems described herein, a variety of detectionmethods and systems are employed, depending upon the specific operationthat is being performed by the system. A microfluidic system can alsoemploy multiple different detection systems for monitoring the output ofthe system. Detection systems of the present invention are used todetect and monitor the materials in the detection window. Once detected,the flow rate and velocity of particles in the channels is optionallymeasured and controlled as described above.

Examples of detection systems include optical sensors, temperaturesensors, pressure sensors, pH sensors, conductivity sensors, and thelike. Each of these types of sensors is readily incorporated into themicrofluidic systems described herein. In these systems, such detectorsare placed either within or adjacent to the microfluidic device or oneor more channels, chambers or conduits of the device, such that thedetector is within sensory communication with the device, channel, orchamber. The phrase “within sensory communication” of a particularregion or element, as used herein, generally refers to the placement ofthe detector in a position such that the detector is capable ofdetecting the property of the microfluidic device, a portion of themicrofluidic device, or the contents of a portion of the microfluidicdevice, for which that detector was intended. For example, a pH sensorplaced in sensory communication with a microscale channel is capable ofdetermining the pH of a fluid disposed in that channel. Similarly, atemperature sensor placed in sensory communication with the body of amicrofluidic device is capable of determining the temperature of thedevice itself.

Particularly preferred detection systems include optical detectionsystems for detecting an optical property of a material within thechannels and/or chambers of the microfluidic devices that areincorporated into the microfluidic systems described herein. Suchoptical detection systems are typically placed adjacent to a microscalechannel of a microfluidic device, and are in sensory communication withthe channel via an optical detection window that is disposed across thechannel or chamber of the device. Optical detection systems includesystems that are capable of measuring the light emitted from materialwithin the channel, the transmissivity or absorbance of the material, aswell as the materials spectral characteristics. In preferred aspects,the detector measures an amount of light emitted from the material, suchas a fluorescent or chemiluminescent material. As such, the detectionsystem will typically include collection optics for gathering a lightbased signal transmitted through the detection window, and transmittingthat signal to an appropriate light detector. Microscope objectives ofvarying power, field diameter, and focal length are readily utilized asat least a portion of this optical train. The light detectors areoptionally photodiodes, avalanche photodiodes, photomultiplier tubes,diode arrays, or in some cases, imaging systems, such as charged coupleddevices (CCDs) and the like. In preferred aspects, photodiodes areutilized, at least in part, as the light detectors. The detection systemis typically coupled to a computer (described in greater detail below),via an analog to digital or digital to analog converter, fortransmitting detected light data to the computer for analysis, storageand data manipulation.

In the case of fluorescent materials, the detector will typicallyinclude a light source which produces light at an appropriate wavelengthfor activating the fluorescent material, as well as optics for directingthe light source through the detection window to the material containedin the channel or chamber. The light source can be any number of lightsources that provides an appropriate wavelength, including lasers, laserdiodes and LEDs. Other light sources required for other detectionsystems. For example, broad band light sources are typically used inlight scattering/transmissivity detection schemes, and the like.Typically, light selection parameters are well known to those of skillin the art.

The detector can exist as a separate unit, but is preferably integratedwith the controller system, into a single instrument. Integration ofthese functions into a single unit facilitates connection of theseinstruments with the computer (described below), by permitting the useof few or a single communication port(s) for transmitting informationbetween the controller, the detector and the computer.

Computer

As noted above, either or both of the controller system and/or thedetection system are coupled to an appropriately programmed processor orcomputer which functions to instruct the operation of these instrumentsin accordance with preprogrammed or user input instructions, receivedata and information from these instruments, and interpret, manipulateand report this information to the user. As such, the computer istypically appropriately coupled to one or both of these instruments(e.g., including an analog to digital or digital to analog converter asneeded).

The computer typically includes appropriate software for receiving userinstructions, either in the form of user input into a set parameterfields, e.g., in a GUI, or in the form of preprogrammed instructions,e.g., preprogrammed for a variety of different specific operations. Thesoftware then converts these instructions to appropriate language forinstructing the operation of the fluid direction and transportcontroller to carry out the desired operation. The computer thenreceives the data from the one or more sensors/detectors included withinthe system, and interprets the data, either provides it in a userunderstood format, or uses that data to initiate further controllerinstructions, in accordance with the programming, e.g., such as inmonitoring and control of flow rates, temperatures, applied voltages,and the like.

In the present invention, the computer typically includes software forthe monitoring of materials in the channels, so that flow rate andvelocity may be determined. Additionally the software is optionally usedto control electrokinetic injection or withdrawal of material. Theelectrokinetic or withdrawal is used to modulate the flow rate asdescribed above.

Kits

Generally, the microfluidic devices described herein are packaged toinclude many if not all of the necessary reagents for performing thedevice's preferred function. For example, the kits can include any ofmicrofluidic devices comprising arrays, particle array members, reagents(e.g., sequencing or PCR reagents), sample materials, control materials,or the like. Such kits also typically include appropriate instructionsfor using the devices and reagents, and in cases where reagents are notpredisposed in the devices themselves, with appropriate instructions forintroducing the reagents into the channels and/or chambers of thedevice. In this latter case, these kits optionally include specialancillary devices for introducing materials into the microfluidicsystems, e.g., appropriately configured syringes/pumps, or the like (ofcourse, in one preferred embodiment, the device itself comprises apipettor element, such as an electropipettor for introducing materialinto channels and chambers within the device). In the former case, suchkits typically include a microfluidic device with necessary reagentspredisposed in the channels/chambers of the device. Generally, suchreagents are provided in a stabilized form, so as to prevent degradationor other loss during prolonged storage, e.g., from leakage. A number ofstabilizing processes are widely used for reagents that are to bestored, such as the inclusion of chemical stabilizers (i.e., enzymaticinhibitors, microcides/bacteriostats, anticoagulants), the physicalstabilization of the material, e.g., through immobilization on a solidsupport, entrapment in a matrix (i.e., a gel), lyophilization, or thelike.

The discussion above is generally applicable to the aspects andembodiments of the invention described below.

Moreover, modifications can be made to the method and apparatusdescribed herein without departing from the spirit and scope of theinvention as claimed, and the invention can be put to a number ofdifferent uses including the following:

The use of a microfluidic system containing at least a first substrateand having a first channel and a second channel intersecting the firstchannel, at least one of the channels having at least onecross-sectional dimension in a range from 0.1 to 500 μm, in order totest the effect of each of a plurality of test compounds on abiochemical system, the system including an array.

The use of a microfluidic system as described herein, wherein abiochemical system flows through one of said channels substantiallycontinuously, providing for, e.g., sequential testing of a plurality oftest compounds.

The use of an array in a microfluidic device as described herein tomodulate reactions within microchannels or microchambers.

The use of electrokinetic injection in a microfluidic device asdescribed herein to modulate or achieve flow in the channels.

The use of a combination of wicks, electrokinetic injection and pressurebased flow elements in a microfluidic device as described herein tomodulate or achieve flow of materials to arrays, or array members tomaterials, e.g., in the channels of the device.

The use of split waste wells or split reagent wells to load or unloadreagents from a microfluidic device as described herein.

An assay utilizing a use of any one of the microfluidic systems orsubstrates described herein.

Microfluidic devices and bioassays which can be adapted to the presentinvention by the addition of arrays include various PCT applications andissued U.S. patents, such as, U.S. Pat. No. 5,699,157 (J. Wallace Parce)issued Dec. 16, 1997, U.S. Pat. No. 5,779,868 (J. Wallace Parce et al.)issued Jul. 14, 1998, U.S. Pat. No. 5,800,690 (Calvin Y. H. Chow et al.)issued Sep. 1, 1998, and U.S. Pat. No. 5,842,787 (Anne R. Kopf-Sill etal.) issued Dec. 1, 1998; and published PCT applications, such as, WO98/00231, WO 98/00705, WO 98/00707, WO 98/02728, WO 98/05424, WO98/22811, WO 98/45481, WO 98/45929, WO 98/46438, and WO 98/49548, whichare all incorporated herein by reference.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovemay be used in various combinations. All publications and patentdocuments cited in this application are incorporated by reference intheir entirety for all purposes to the same extent as if each individualpublication or patent document were so individually denoted.

What is claimed is:
 1. A method of performing a plurality of chemicalreactions in a microfluidic device, the method comprising: providing amicrofluidic device comprising an interior cavity, the cavity comprisinga main channel and first and second side channels, the first and secondside channels intersecting the main channel, thereby forming first andsecond channel intersections, the cavity further comprising first andsecond wells, the first well being in fluid communication with the firstside channel and the second well being in fluid communication with thesecond side channel; providing a mobile ordered array comprising first,second, and third sets of particles within the main channel of theinterior cavity; providing first and second liquid reagents within theinterior cavity, the first liquid reagent being disposed within thefirst well and the second liquid reagent being disposed within thesecond well; transporting the first set of particles as the mobileordered array through the main channel into the first channelintersection by fluid pressure; flowing the first liquid reagent fromthe first well through the first side channel into the first channelintersection, wherein the first liquid reagent contacts the first set ofparticles in the main channel, thereby providing a first chemicalreaction in the microfluidic device; transporting the first set ofparticles as the mobile ordered array through the main channel into thesecond channel intersection; and flowing the second liquid reagent fromthe second well through the second side channel into the second channelintersection, wherein the second liquid reagent contacts the first setof particles in the main channel, thereby providing a second chemicalreaction in the microfluidic device; wherein the particles are beads. 2.The method of claim 1, the method further comprising: transporting thesecond set of particles through the main channel into the first channelintersection; and flowing the first liquid reagent through the firstside channel into the first channel intersection, wherein the firstreagent contacts the second set of particles in the main channel.
 3. Themethod of claim 1, the method further comprising detecting a signalresulting from contact of the first set of particles by one of the firstand the second liquid reagents.
 4. The method of claim 1, the methodfurther comprising detecting a plurality of signals resulting fromcontact of the first set of particles by the first and the second liquidreagents.
 5. The method of claim 2, the method further comprisingdetecting a plurality of signals resulting from contact of the first andsecond sets of particles by the first liquid reagent.
 6. The method ofclaim 5, wherein detecting a plurality of signals resulting from contactof the first and second sets of particles by the first liquid reagentcomprises detecting simultaneous or sequential signal emissions from thefirst and second sets of particles.
 7. The method of claim 1, wherein atleast one of the first and second liquid reagents is a nucleic acidsequencing reagent selected from the group consisting of a liquidsolution comprising a nucleotide, a liquid solution comprising apolymerase, a liquid solution comprising a dNTP or ddNTP, a liquidsolution comprising a fluorescently labeled nucleotide, a liquidsolution comprising a nucleotide analog, a liquid solution comprising asufurylase, a liquid solution comprising an apyrase, a liquid solutioncomprising inorganic phosphate, a liquid solution comprising ATP, aliquid solution comprising a thermostable polymerase, a liquid solutioncomprising an endonuclease, a liquid solution comprising an exonuclease,a liquid solution comprising Mg++, a liquid solution comprising amolecular crowding agent, a liquid solution comprising a buffer, aliquid solution comprising an intercalating dye, a liquid solutioncomprising a reducing agent, a liquid solution comprising a phosphataseor an enzyme with phosphatase activity, a liquid solution comprising asalt, and combinations thereof.
 8. The method of claim 1, wherein atleast one of the first and second liquid reagents is a binding reagentselected from the group consisting of a first nucleic acid that is fullyor partially complementary to a second nucleic acid complexed with atleast one of the first and second particle sets, a first protein thatspecifically binds to one or more component of at least one of the firstand second particle sets, a first antibody that specifically binds toone or more component of at least one of the first and second particlesets, a binding buffer, a blocking reagent, an immunoassay reagent, alabeled probe nucleic acid, and combinations thereof; and wherein themethod further comprises flowing the at least one of the first andsecond liquid reagents into contact with at least one of the first andsecond sets of particles and detecting resulting binding of the liquidreagent to the at least one of the first and second sets of particles.9. The method of claim 1, wherein at least one of the first and secondsets of particles comprises at least one set of nucleic acid templatesand at least one set of nucleic acid primers, wherein at least one ofthe first and second liquid reagents comprises one or more nucleotideanalogs, and wherein at least one of the first and the second chemicalreactions comprises adding at least one of the one or more nucleotideanalogs to at least one member of the set of primers.
 10. The method ofclaim 9, wherein the one or more nucleotide analogs comprise reversiblechain-terminating nucleotide analogs.
 11. The method of claim 10,wherein the reversible chain-terminating nucleotide analogs comprise acompound having formula (I) or formula (II), and wherein B comprises anitrogenous base and R⁴ comprises a label moiety:


12. The method of claim 1, wherein at least one of the first and secondsets of particles comprises at least one set of nucleic acid templatesand at least one set of nucleic acid primers, wherein at least one ofthe liquid reagents comprises one or more nucleotides, and wherein atleast one of the first and second chemical reactions results in adouble-stranded nucleic acid region, the method further comprisingflowing an intercalating dye into contact with the double-strandednucleic acid region, which intercalating dye intercalates into thedouble-stranded region.
 13. The method of claim 1, further comprisingadding an intercalating dye to a nucleic acid complementary to atemplate molecule bound to one or more of the first and second sets ofparticles, the method further comprising detecting the intercalatingdye.
 14. The method of claim 1, the cavity further comprising third andfourth side channels, the third and fourth side channels intersectingthe main channel, thereby forming third and fourth channelintersections, the method further comprising: providing third and fourthliquid reagents, wherein each of the first, second, third, and fourthliquid reagents comprises one of A, G, C, or T nucleotides; transportingthe first set of particles through each of the third and fourth channelintersections; flowing the third reagent through the third side channelinto the third channel intersection; and flowing the fourth reagentthrough the fourth side channel into the fourth channel intersection,wherein each of the third and fourth liquid reagents contacts the firstset of particles in the main channel.
 15. The method of claim 2, thecavity further comprising third and fourth side channels, the third andfourth side channels intersecting the main channel, thereby formingthird and fourth channel intersections, the method further comprising:providing third and fourth liquid reagents, wherein each of the first,second, third, and fourth liquid reagents comprises one of A, G, C, or Tnucleotides; transporting the second set of particles through each ofthe second, third, and fourth channel intersections; flowing the secondreagent through the second side channel into the third channelintersection; flowing the third reagent through the third side channelinto the third channel intersection; and flowing the fourth reagentthrough the fourth side channel into the fourth channel intersection,wherein each of the second, third, and fourth liquid reagents contactsthe second set of particles in the main channel.
 16. The method of claim1, further comprising sequencing by synthesis, the sequencing bysynthesis method comprising addition of an intercalating dye to anucleic acid complementary to a template molecule bound to a member ofthe mobile ordered array, the method further comprising detecting theintercalating dye.
 17. The method of claim 1, further comprisingsequencing by synthesis, the method comprising addition of a chainterminator to a nucleic acid complementary to a template molecule boundto a member of the mobile ordered array, the chain terminator comprisinga label.
 18. The method of claim 1, further comprising sequencing bysynthesis, the method comprising addition of a reversible chainterminator to a nucleic acid complementary to a template molecule boundto a member of the mobile ordered array, the reversible chain terminatorcomprising a label, and the method further comprising cleavage of thereversible chain terminator.